Cell carrier and cell carrier containment devices containing regenerative cells

ABSTRACT

The present invention relates to a device comprising a cell carrier portion containing regenerative cells, e.g., stem and progenitor cells, and a cell carrier containment portion. The device is useful for the treatment of bone related disorders, including spinal fusion related disorders and long bone or flat bone related defects. The device may be used in conjunction with disclosed automated systems and methods for separating and concentrating regenerative cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/568,533, filed Sep. 28, 2009 entitled CELL CARRIER AND CELL CARRIERCONTAINMENT DEVICES, which is a divisional of U.S. patent applicationSer. No. 10/885,293, now U.S. Pat. No. 7,595,043, filed on Jul. 1, 2004,entitled CELL CARRIER AND CELL CARRIER CONTAINMENT DEVICES CONTAININGREGENERATIVE CELLS, which is a continuation-in-part application of U.S.patent application Ser. No. 10/316,127, filed on Dec. 9, 2002, entitledSYSTEMS AND METHODS FOR TREATING PATIENTS WITH PROCESSED LIPOASPIRATECELLS, now abandoned, which claims the benefit of U.S. ProvisionalApplication No. 60/338,856, filed Dec. 7, 2001. U.S. patent applicationSer. No. 10/885,293, now U.S. Pat. No. 7,595,043, also claims priorityto U.S. Provisional Application No. 60/554,455, entitled CELL CARRIERAND CELL CARRIER CONTAINMENT DEVICES CONTAINING ADIPOSE DERIVED CELLS,filed Mar. 19, 2004. The contents of each of the aforementionedapplications are expressly incorporated herein by this reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods of making and using a device,comprised of a cell carrier portion and a cell carrier containmentportion, in combination with regenerative cells, e.g., adipose derivedregenerative cells, for the treatment of bone related disorders. Inparticular, the novel combination disclosed herein is useful forpromoting bone and/or cartilage formation. The present invention isespecially useful for promoting bone formation in normallynon-osteoconductive areas, e.g., for disorders requiring therapy in theform of interbody spinal fusion surgery.

2. Description of the Related Art

Back pain caused by disc degeneration and pressure on nerves near thespine affects nearly 65 million Americans. Common early treatmentoptions include stretching exercises, pain medications or steroidinjections into back muscles. If these treatments fail, spinal fusionsurgery becomes an option. According to the American Academy ofOrthopedic Surgeons, about 250,000 spinal fusion surgeries are performedevery year, mostly on adults between the ages of 45 to 64.

Spinal fusion is a process by which two or more of the vertebrae thatmake up the spinal column are fused together with bone grafts andinternal devices (such as rods) that heal into a single solid bone.Spinal fusion can eliminate unnatural motion between the vertebrae and,in turn, reduce pressure on nerve endings. In addition, spinal fusioncan be used to treat, for example, injuries to spinal vertebrae causedby trauma; protrusion and degeneration of the cushioning disc betweenvertebrae (sometimes called slipped disc or herniated disc); abnormalcurvatures (such as scoliosis or kyphosis); and weak or unstable spinecaused by infections or tumors.

Autogenous bone (bone from the patient) or allograft bone (bone fromanother individual) are the most commonly used materials to induce boneformation. Generally, small pieces of bone are placed into the spacebetween the vertebrae to be fused. Sometimes larger solid pieces of boneare used to provide immediate structural support. Autogenous bone isgenerally considered superior at promoting fusion. However, thisprocedure requires extra surgery to remove bone from another area of thepatient's body such as the pelvis or fibula. Thus, it has been reportedthat about 30 percent of patients have significant pain and tendernessat the graft harvest site, which may be prolonged, and in some casesoutlast the back pain the procedure intended to correct. Similarly,allograft bone and other bone graft substitutes, although eliminatingthe need for a second surgery, have drawbacks in that they have yet tobe proven as cost effective and efficacious substitutes for autogenousbone fusion.

An alternative to autogenous or allograft bone is the use of growthfactors that promote bone formation. For example, studies have shownthat the use of bone morphogenic proteins (“BMPs”) results in betteroverall fusion, less time in the operating room and, more importantly,fewer complications for patients because it eliminates the need for thesecond surgery. However, use of BMPs, although efficacious in promotingbone growth, can be prohibitively expensive.

Another alternative is the use of a genetically engineered version of anaturally occurring bone growth factor. This approach also haslimitations. Specifically, surgeons have expressed concerns thatgenetically engineered BMPs can dramatically speed the growth ofcancerous cells or cause non-cancerous cells to become more sinister.Another concern is unwanted bone creation. There is a chance that bonegenerated by genetically engineered BMPs could form over the delicatenerve endings in the spine or, worse, somewhere else in the body.

Regenerative medicine, which harnesses the ability of regenerativecells, e.g., stem cells (i.e., the unspecialized master cells of thebody) to renew themselves indefinitely and develop into maturespecialized cells, may be a means of circumventing the limitations ofthe prior-art techniques. Stem cells, i.e., both embryonic and adultstem cells, have been shown to possess the nascent capacity to becomemany, if not all, of the 200+ cell and tissue types of the body,including bone. Recently, adipose tissue has been shown to be a sourceof adult stem cells (Zuk et al., 2001; Zuk et al., 2002). Adipose tissue(unlike marrow, skin, muscle, liver and brain) is comparably easy toharvest in relatively large amounts with low morbidity (Commons et al.,2001; Katz et al., 2001b). Accordingly, given the limitations of theprior art spinal fusion techniques, there exists a need for a devicethat incorporates regenerative cells, e.g., stem cells that posses theability to induce bone formation.

SUMMARY OF THE INVENTION

The present invention relates to methods of making and using a devicecomprised of a cell carrier portion and a cell carrier containmentportion, and a therapeutically effective dose of regenerative cells, thecombination of which promotes bone and/or cartilage formation in anintended area for bone formation in the recipient, i.e., a target area.The device and cells of the present invention are especially useful fortreating disorders that benefit from bone formation in normallynon-osteoconductive areas, e.g., for disorders requiring therapy in theform of interbody spinal fusion surgery. The device and cells of thepresent invention are also useful for treating other disorders ordefects that cannot heal by osteoconduction, e.g., defects in long bonesas a result of trauma. In a preferred embodiment, one or more portionsof the device is resorbable.

The present invention further relates to systems and methods forpreparing regenerative cells, e.g., adult stem and progenitor cells. Thepresent invention is yet further based on the discovery of methods andcompositions of regenerative cells, e.g., adult stem and progenitorcells, that promote bone and/or cartilage formation. Accordingly, in oneembodiment, the present invention is directed to compositions, methods,and systems for using cells derived from adipose tissue that are placedinto the cell carrier portion of the device along with such additivesnecessary to promote, engender, or support bone and/or cartilageformation in a recipient.

In one embodiment, the entire procedure from tissue extraction throughprocessing and placement of the device into the intended bone and/orcartilage formation site of the recipient would all be performed in thesame facility, indeed, even within the same room of the patientundergoing the procedure. In a particular embodiment, an effective doseof the regenerative cells, e.g., stem and progenitor cells, are placedonto the cell carrier portion of the device prior to insertion in thetarget area. In another embodiment, an effective dose of theregenerative cells, e.g., stem and progenitor cells, are placed onto thecell carrier subsequent to placement of the device into the intendedbone and/or cartilage formation site of the recipient, e.g., via asyringe. In yet other embodiments, the cells may be placed into the cellcarrier portion of the device in combination with other cells, tissue,tissue fragments, or other stimulators of cell growth and/ordifferentiation. In a preferred embodiment, the cells, with any of theabove mentioned additives, are placed into the person (via the device)from whom they were obtained. In a preferred embodiment, the cellcarrier portion and/or the cell carrier containment portion isresorbable.

In a certain embodiment, promoting bone and/or cartilage formation in apatient includes steps of: a) providing a tissue removal system; b)removing adipose tissue from a patient using the tissue removal system,the adipose tissue having a concentration of stem cells; c) processingat least a part of the adipose tissue to obtain a concentration of stemcells sufficient to constitute an effective dose for promoting boneand/or cartilage formation; d) adding the regenerative cells, e.g., stemand progenitor cells, obtained from the processing step to a cellcarrier portion of the device of the invention; and e) inserting thedevice containing the cells into an intended bone and/or cartilageformation site of the recipient. In a preferred embodiment, the cellcarrier portion and/or the cell carrier containment portion isresorbable. In another preferred embodiment, the tissue processingsystem is automated.

In another embodiment, promoting bone and/or cartilage formation in apatient includes steps of: a) providing a tissue removal system; b)removing adipose tissue from a patient using the tissue removal system,the adipose tissue having a concentration of stem cells; c) processingat least a part of the adipose tissue to obtain a concentration of stemcells sufficient to constitute an effective dose for promoting boneand/or cartilage formation; d) inserting the device of the inventioninto an intended bone and/or cartilage formation site of the recipient;and e) adding the regenerative cells obtained from the processing stepto the cell carrier portion of the device inserted into the recipient.In a preferred embodiment, the cell carrier portion and/or the cellcarrier containment portion is resorbable. In another preferredembodiment, the tissue processing system is automated.

Any feature or combination of features described herein are includedwithin the scope of the present invention provided that the featuresincluded in any such combination are not mutually inconsistent as willbe apparent from the context, this specification, and the knowledge ofone of ordinary skill in the art. Additional advantages and aspects ofthe present invention are apparent in the following detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. is an illustration of a system for separating and concentratingregenerative cells from tissue which includes one filter assembly.

FIG. 2 is an illustration of a system similar to FIG. 1 having aplurality of filter assemblies in a serial configuration.

FIG. 3 is an illustration of a system similar to FIG. 1 having aplurality of filter assemblies in a parallel configuration.

FIG. 4 is an illustration of a system for separating and concentratingregenerative cells from tissue which includes a centrifuge chamber.

FIG. 5 is a sectional view of a collection chamber including a prefixedfilter utilized in a system for separating and concentratingregenerative cells from tissue.

FIG. 6 is a sectional view of a processing chamber of a system forseparating and concentrating regenerative cells from tissue utilizing apercolative filtration system.

FIG. 7 is a sectional view of a processing chamber of a system forseparating and concentrating regenerative cells utilizing a centrifugedevice for concentrating the regenerative cells.

FIG. 8 is another sectional view of the processing chamber of FIG. 7.

FIGS. 9A, 9B and 9C illustrate an elutriation component in use with thesystem of the invention.

FIG. 10 is an illustration of a system for separating and concentratingregenerative cells from tissue utilizing vacuum pressure to move fluidsthrough the system. A vacuum system can be constructed by applying avacuum pump or vacuum source to the outlet of the system, controlled ata predetermined rate to pull tissue and fluid through, using a system ofstopcocks, vents, and clamps to control the direction and timing of theflow.

FIG. 11 is an illustration of a system for separating and concentratingregenerative cells from tissue utilizing positive pressure to movefluids through the system. A positive pressure system uses a mechanicalmeans such as a peristaltic pump to push or propel the fluid and tissuethrough the system at a determined rate, using valves, stopcocks, vents,and clamps to control the direction and timing of the flow.

FIG. 12A illustrates a filtration process in which the feed stream offluid flows tangentially to the pores of the filter. FIG. 12Billustrates a filtration process in which the feed stream of fluid flowsperpendicular to the pores of the filter.

FIG. 13 is an illustration of an exemplary disposable set for a systemof the invention.

FIGS. 14-1 and 14-2 are illustrations of an exemplary re-usablecomponent for a system of the invention.

FIG. 15A-1 and 15A-2 are illustrations of an exemplary device of theinvention assembled using the disposable set of FIG. 13 and a re-usablecomponent of FIG. 14.

FIG. 15B is a flowchart depicting exemplary pre-programmed steps,implemented through a software program, that control automatedembodiments of a system of the present invention. Two alternativeprocessing parameters are shown indicating the versatility of thesystem.

FIG. 16 depicts a three-dimensional view of an exemplary cell carrierportion of the device of the invention.

FIG. 17 depicts the cell carrier of FIG. 16 seeded with the regenerativecells of the present invention.

FIG. 18 depicts a three-dimensional view of an exemplary containmentportion of the device of the invention.

FIG. 19 depicts a three-dimensional view of an exemplary device of theinvention.

FIG. 20 depicts a three-dimensional view of an exemplary combination ofthe device and adipose-derived cells of the invention.

FIG. 21 depicts a three-dimensional view of another exemplarycontainment portion of the device of the invention.

FIG. 22 depicts a three-dimensional view of another exemplarycontainment portion of the device of the invention.

FIG. 23 depicts a three-dimensional view of another exemplarycontainment portion of the device of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a minimally invasive and cost-effectivedevice for promoting bone and/or cartilage formation using atherapeutically effective dose of regenerative cells. The presentinvention is based, in part, on the discovery that the regenerativecells of the invention are a rich source of bone and cartilageprogenitor cells, e.g., osteoprogenitor cells. Furthermore, theregenerative cells of the invention have been shown herein to form boneand cartilage in vivo. Accordingly, the device of the present inventioncan be used to treat bone related disorders, e.g., by promoting boneand/or cartilage formation. Specifically, the present invention providesa novel combination of 1) a device, comprising a porous cell carrierportion, and a cell carrier containment portion (hereinafter referred toherein as “the device”), and 2) a therapeutically effective dose ofadipose derived regenerative cells, e.g., adult stem and progenitorcells, that promote bone and/or cartilage formation. In a particularembodiment, the device and cells of the present invention are useful fortreating disorders requiring bone formation in normallynon-osteoconductive areas, e.g., for the treatment of disordersrequiring therapy in the form of interbody spinal fusion surgery and thelike.

The present invention also relates to rapid and reliable systems andmethods for separating and concentrating regenerative cells, e.g., stemcells and/or progenitor cells, from a wide variety of tissues, includingbut not limited to, adipose, bone marrow, blood, skin, muscle, liver,connective tissue, fascia, brain and other nervous system tissues, bloodvessels, and other soft or liquid tissues or tissue components or tissuemixtures (e.g., a mixture of tissues including skin, blood vessels,adipose, and connective tissue). In a preferred embodiment, the systemseparates and concentrates regenerative cells from adipose tissue. Inanother preferred embodiment, the system is automated such that theentire method may be performed with minimal user intervention orexpertise. In a particularly preferred embodiment, the regenerativecells obtained using the systems and methods of the present inventionare suitable for direct placement into a recipient suffering from arenal disease or disorder from whom the tissue was extracted.

Preferably, the entire procedure from tissue extraction throughseparating, concentrating and placement of the regenerative cells intothe recipient would all be performed in the same facility, indeed, evenwithin the same room of the patient undergoing the procedure. Theregenerative cells may be used in a relatively short time period afterextraction and concentration. For example, the regenerative cells may beready for use in about one hour from the harvesting of tissue from apatient, and in certain situations, may be ready for use in about 10 to40 minutes from the harvesting of the tissue. In a preferred embodiment,the regenerative cells may be ready to use in about 20 minutes from theharvesting of tissue. The entire length of the procedure from extractionthrough separating and concentrating may vary depending on a number offactors, including patient profile, type of tissue being harvested andthe amount of regenerative cells required for a given therapeuticapplication. The cells may also be placed into the recipient incombination with other cells, tissue, tissue fragments, scaffolds orother stimulators of cell growth and/or differentiation in the contextof a single operative procedure with the intention of deriving atherapeutic, structural, or cosmetic benefit to the recipient. It isunderstood that any further manipulation of the regenerative cellsbeyond the separating and concentrating phase of the system will requireadditional time commensurate with the manner of such manipulation.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

As used herein, “regenerative cells” refers to any heterogeneous orhomologous cells obtained using the systems and methods of the presentinvention which cause or contribute to complete or partial regeneration,restoration, or substitution of structure or function of an organ,tissue, or physiologic unit or system to thereby provide a therapeutic,structural or cosmetic benefit. Examples of regenerative cells include:ASCs, endothelial cells, endothelial precursor cells, endothelialprogenitor cells, macrophages, fibroblasts, pericytes, smooth musclecells, preadipocytes, differentiated or de-differentiated adipocytes,keratinocytes, unipotent and multipotent progenitor and precursor cells(and their progeny), and lymphocytes.

One mechanism by which the regenerative cells may provide a therapeutic,structural or cosmetic benefit is by incorporating themselves or theirprogeny into newly generated, existing or repaired tissues or tissuecomponents. For example, ASCs and/or their progeny may incorporate intonewly generated bone, muscle, or other structural or functional tissueand thereby cause or contribute to a therapeutic, structural or cosmeticimprovement. Similarly, endothelial cells or endothelial precursor orprogenitor cells and their progeny may incorporate into existing, newlygenerated, repaired, or expanded blood vessels to thereby cause orcontribute to a therapeutic, structural or cosmetic benefit. Anothermechanism by which the regenerative cells may provide a therapeutic,structural or cosmetic benefit is by expressing and/or secretingmolecules, e.g., growth factors, that promote creation, retention,restoration, and/or regeneration of structure or function of a giventissue or tissue component.

The regenerative cells may be used in their ‘native’ form as present inor separated and concentrated from the tissue using the systems andmethods of the present invention or they may be modified by stimulationor priming with growth factors or other biologic response modifiers, bygene transfer (transient or stable transfer), by furthersub-fractionation of the resultant population on the basis or physicalproperties (for example size or density), differential adherence to asolid phase material, expression of cell surface or intracellularmolecules, cell culture or other ex vivo or in vivo manipulation,modification, or fractionation as further described herein. Theregenerative cells may also be used in combination with other cells ordevices such as synthetic or biologic scaffolds, materials or devicesthat deliver factors, drugs, chemicals or other agents that modify orenhance the relevant characteristics of the cells as further describedherein.

As used herein, “regenerative cell composition” refers to thecomposition of cells typically present in a volume of liquid after atissue, e.g., adipose tissue, is washed and at least partiallydisaggregated. For example, a regenerative cell composition of theinvention comprises multiple different types of regenerative cells,including ASCs, endothelial cells, endothelial precursor cells,endothelial progenitor cells, macrophages, fibroblasts, pericytes,smooth muscle cells, preadipocytes, differentiated or de-differentiatedadipocytes, keratinocytes, unipotent and multipotent progenitor andprecursor cells (and their progeny), and lymphocytes. The regenerativecell composition may also contain one or more contaminants, such ascollagen, which may be present in the tissue fragments, or residualcollagenase or other enzyme or agent employed in or resulting from thetissue disaggregation process described herein.

As used herein, “regenerative medicine” refers to any therapeutic,structural or cosmetic benefit that is derived from the placement,either directly or indirectly, of regenerative cells into a subject. Asused herein, the phrase “bone related disorder” is intended to includeall bone related disorders, including, for example, disorders requiringspinal fixation, spinal stabilization, repair of segmental defects inthe body (such as in long bones and flat bones) and growth of bone in anormally non-osteoconductive environment are bone related disorders. Ina particular embodiment, the phrase is intended to include disordersrequiring spinal fusion surgery. In addition, bone related disorders isintended to encompass disorders of the vertebrae and discs including,but are not limited to, disruption of the disc annulus such as annularfissures, chronic inflammation of the disc, localized disc herniationswith contained or escaped extrusions, relative instability of thevertebrae surrounding the disc and degenerative disc disease. Treatmentof a bone related disease or disorder is within the ambit ofregenerative medicine.

As used herein, “stem cell” refers to a multipotent regenerative cellwith the potential to differentiate into a variety of other cell types,which perform one or more specific functions and have the ability toself-renew. Some of the stem cells disclosed herein may be multipotent.

As used herein, “progenitor cell” refers to a multipotent regenerativecell with the potential to differentiate into more than one cell typeand has limited or no ability to self-renew. “Progenitor cell”, as usedherein, also refers to a unipotent cell with the potential todifferentiate into only a single cell type, which performs one or morespecific functions and has limited or no ability to self-renew. Inparticular, as used herein, “endothelial progenitor cell” refers to amultipotent or unipotent cell with the potential to differentiate intovascular endothelial cells.

As used herein, “precursor cell” refers to a unipotent regenerative cellwith the potential to differentiate into one cell type. Precursor cellsand their progeny may retain extensive proliferative capacity, e.g.,lymphocytes and endothelial cells, which can proliferate underappropriate conditions.

As used herein “stem cell number” or “stem cell frequency” refers to thenumber of colonies observed in a clonogenic assay in which adiposederived cells (ADC) are plated at low cell density (<10,000 cells/well)and grown in growth medium supporting MSC growth (for example, DMEM/F12medium supplemented with 10% fetal calf serum, 5% horse serum, andantibiotic/antimycotic agents). Cells are grown for two weeks afterwhich cultures are stained with hematoxylin and colonies of more than 50cells are counted as CFU-F. Stem cell frequency is calculated as thenumber of CFU-F observed per 100 nucleated cells plated (for example; 15colonies counted in a plate initiated with 1,000 nucleated regenerativecells gives a stem cell frequency of 1.5%). Stem cell number iscalculated as stem cell frequency multiplied by the total number ofnucleated ADC cells obtained. A high percentage (˜100%) of CFU-F grownfrom regenerative cells express the cell surface molecule CD105 which isalso expressed by marrow-derived stem cells (Barry et al., 1999). CD105is also expressed by adipose tissue-derived stem cells (Zuk et al.,2002).

As used herein, the term “adipose tissue” refers to fat including theconnective tissue that stores fat. Adipose tissue contains multipleregenerative cell types, including ASCs and endothelial progenitor andprecursor cells.

As used herein, the term “unit of adipose tissue” refers to a discreteor measurable amount of adipose tissue. A unit of adipose tissue may bemeasured by determining the weight and/or volume of the unit. Based onthe data identified above, a unit of processed lipoaspirate, as removedfrom a patient, has a cellular component in which at least 0.1% of thecellular component is stem cells; that is, it has a stem cell frequency,determined as described above, of at least 0.1%. In reference to thedisclosure herein, a unit of adipose tissue may refer to the entireamount of adipose tissue removed from a patient, or an amount that isless than the entire amount of adipose tissue removed from a patient.Thus, a unit of adipose tissue may be combined with another unit ofadipose tissue to form a unit of adipose tissue that has a weight orvolume that is the sum of the individual units.

As used herein, the term “portion” refers to an amount of a materialthat is less than a whole. A minor portion refers to an amount that isless than 50%, and a major portion refers to an amount greater than 50%.Thus, a unit of adipose tissue that is less than the entire amount ofadipose tissue removed from a patient is a portion of the removedadipose tissue.

As used herein, the term “processed lipoaspirate” refers to adiposetissue that has been processed to separate the active cellular component(e.g., the component containing regenerative) from the mature adipocytesand connective tissue. This fraction is referred to herein as“adipose-derived cells” or “ADC.” Typically, ADC refers to the pellet ofregenerative cells obtained by washing and separating and concentratingthe cells from the adipose tissue. The pellet is typically obtained bycentrifuging a suspension of cells so that the cells aggregate at thebottom of a centrifuge chamber or cell concentrator.

As used herein, the terms “administering,” “introducing,” “delivering,”“placement” and “transplanting” are used interchangeably herein andrefer to the placement of the regenerative cells of the invention into asubject by a method or route which results in at least partiallocalization of the regenerative cells at a desired site. Theregenerative cells can be administered by any appropriate route whichresults in delivery to a desired location in the subject where at leasta portion of the cells or components of the cells remain viable. Theperiod of viability of the cells after administration to a subject canbe as short as a few hours, e.g., twenty-four hours, to a few days, toas long as several years.

As used herein, the term “treating” includes reducing or alleviating atleast one adverse effect or symptom of a disease or disorder

As used herein, “therapeutically effective dose of regenerative cells”refers to an amount of regenerative cells that are sufficient to bringabout a beneficial or desired clinical effect. Said dose could beadministered in one or more administrations. However, the precisedetermination of what would be considered an effective dose may be basedon factors individual to each patient, including, but not limited to,the patient's age, size, type or extent of disease, stage of thedisease, route of administration of the regenerative cells, the type orextent of supplemental therapy used, ongoing disease process and type oftreatment desired (e.g., aggressive vs. conventional treatment).

As used herein, the term “subject” includes warm-blooded animals,preferably mammals, including humans. In a preferred embodiment, thesubject is a primate. In an even more preferred embodiment, the subjectis a human.

Reference will now be made in detail to the presently preferredembodiments of the invention, examples of which are illustrated in theaccompanying figures. Wherever possible, the same or similar referencenumbers are used in the drawings and the description to refer to thesame or like parts. It should be noted that the drawings are insimplified form and are not to precise scale. In reference to thedisclosure herein, for purposes of convenience and clarity only,directional terms, such as, top, bottom, left, right, up, down, over,above, below, beneath, rear, and front, are used with respect to theaccompanying drawings. Such directional terms should not be construed tolimit the scope of the invention in any manner.

Although the disclosure herein refers to certain illustratedembodiments, it is to be understood that these embodiments are presentedby way of example and not by way of limitation. The intent of thefollowing detailed description, although discussing exemplaryembodiments, is to be construed to cover all modifications,alternatives, and equivalents of the embodiments as may fall within thespirit and scope of the invention as defined by the appended claims. Thepresent invention may be practiced in conjunction with various cell ortissue separation techniques that are conventionally used in the art,and only so much of the commonly practiced process steps are includedherein as are necessary to provide an understanding of the presentinvention.

I. Methods of the Invention

1. Methods of Obtaining Regenerative Cells (ADC)

As previously set forth herein, regenerative cells, e.g., stem andprogenitor cells, can be harvested from a wide variety of tissues. Thesystem of the present invention may be used for all such tissues.Adipose tissue, however, is an especially rich source of regenerativecells. Accordingly, the system of the present invention is illustratedherein using adipose tissue as a source of regenerative cells by way ofexample only and not limitation.

Adipose tissue can be obtained by any method known to a person ofordinary skill in the art. For example, adipose tissue may be removedfrom a patient by liposuction (syringe or power assisted) or bylipectomy, e.g., suction-assisted lipoplasty, ultrasound-assistedlipoplasty, and excisional lipectomy or combinations thereof. Theadipose tissue is removed and collected and may be processed inaccordance with any of the embodiments of a system of the inventiondescribed herein. The amount of tissue collected depends on numerousfactors, including the body mass index and age of the donor, the timeavailable for collection, the availability of accessible adipose tissueharvest sites, concomitant and pre-existing medications and conditions(such as anticoagulant therapy), and the clinical purpose for which thetissue is being collected. For example, the regenerative cell percentageof 100 ml of adipose tissue extracted from a lean individual is greaterthan that extracted from an obese donor (Table 1). This likely reflectsa dilutive effect of the increased fat content in the obese individual.Therefore, it may be desirable, in accordance with one aspect of theinvention, to obtain larger amounts of tissue from overweight donorscompared to the amounts that would be withdrawn from leaner patients.This observation also indicates that the utility of this invention isnot limited to individuals with large amounts of adipose tissue.

TABLE 1 Effect of Body Mass Index on Tissue and Cell Yield Body MassAmount of Tissue Total Regenerative Index Status Obtained (g) Cell Yield(×10⁷) Normal 641 ± 142 2.1 ± 0.4 Obese 1,225 ± 173   2.4 ± 0.5 p value0.03 0.6

After the adipose tissue is processed, the resulting regenerative cellsare substantially free from mature adipocytes and connective tissue.Accordingly, the system of the present invention generates aheterogeneous plurality of adipose derived regenerative cells which maybe used for research and/or therapeutic purposes. In a preferredembodiment, the cells are suitable for placement or re-infusion withinthe body of a recipient. In other embodiments, the cells may be used forresearch, e.g., the cells can be used to establish stem or progenitorcell lines which can survive for extended periods of time and be usedfor further study.

Referring now to the Figures, a system 10 of the present invention isgenerally comprised of one or more of a tissue collection chamber 20, aprocessing chamber 30, a waste chamber 40, an output chamber 50 and asample chamber 60. The various chambers are coupled together via one ormore conduits 12 such that fluids containing biological material maypass from one chamber to another while maintaining a closed, sterilefluid/tissue pathway. The conduits may comprise rigid or flexible bodiesreferred to interchangeably herein as lumens and tubing, respectively.In certain embodiments, the conduits are in the form of flexible tubing,such as polyethylene tubing conventionally used in clinical settings,silicone or any other material known in the art. The conduits 12 canvary in size depending on whether passage of fluid or tissue is desired.The conduits 12 may also vary in size depending on the amount of tissueor fluid that is cycled through the system. For example, for the passageof fluid, the conduits may have a diameter ranging from about 0.060 toabout 0.750 inches and for the passage of tissue, the conduits may havea diameter ranging from 0.312 to 0.750 inches. Generally, the size ofthe conduits is selected to balance the volume the conduits canaccommodate and the time required to transport the tissue or fluidsthrough said conduits. In automated embodiments of the system, theforegoing parameters, i.e., volume and time for transport, must beidentified such that the appropriate signals can be transmitted to theprocessing device of the system. This allows the device to move accuratevolumes of liquid and tissue from one chamber to another. The flexiletubing used should be capable of withstanding negative pressure toreduce the likelihood of collapse. The flexible tubing used should alsobe capable of withstanding positive pressure which is generated by, forexample, a positive displacement pump, which may be used in the system.

All the chambers of the system may be comprised of one or more ports,e.g., outlet 22 or inlet 21 ports, which accept standard IV, syringe andsuction tubing connections. The ports may be a sealed port such as arubber septum closed syringe needle access port 51. The inlet ports maybe coupled to one or more cannulas (not shown) by way of conduits. Forexample, a tissue inlet port 21 may be coupled to an integrated singleuse liposuction cannula and the conduit may be a flexible tubing. Theconduits are generally positioned to provide fluid passageways from onechamber of the system to another. Towards this end, the conduits andports may be coupled to, for example, a suction device (not shown) whichmay be manually or automatically operated. The suction device may be,e.g., a syringe or an electric pump. The suction device should becapable of providing sufficient negative pressure to aspirate tissuefrom a patient. Generally, any suitable suction device known to one ofordinary skill in the art, e.g., a surgeon, may be used.

The conduits 12 may further comprise one or more clamps (not shown) tocontrol the flow of material among various components of the system. Theclamps are useful for maintaining the sterility of the system byeffectively sealing different regions of the system. Alternatively, theconduits 12 may comprise one or more valves 14 that control the flow ofmaterial through the system. The valves 14 are identified as opencircles in the Figures. In preferred embodiments, the valves may beelectromechanical pinch valves. In another embodiment, the valves may bepneumatic valves. In yet other embodiments, the valves may be hydraulicvalves or mechanical valves. Such valves are preferably activated by acontrol system which may be coupled to levers. The levers may bemanually manipulated such that the levers are activated. In automatedembodiments, the control system may be coupled to the levers as well asto a processing device which may activate the valves at pre-determinedactivation conditions. In certain automated embodiments, activation ofthe valves may be partially automated and partially subject to theuser's preference such that the process may be optimized. In yet otherembodiments, certain valves may be activated manually and othersautomatically through the processing device. The valves 14 may also beused in conjunction with one or more pumps, e.g., peristaltic pumps 34or positive displacement pumps (not shown). The conduits 12 and/or thevalves 14 may also be comprised of sensors 29, e.g., optical sensors,ultrasonic sensors, pressure sensors or other forms of monitors known inthe art that are capable of distinguishing among the various fluidcomponents and fluid levels that flow through the system. In a preferredembodiment, the sensors 29 may be optical sensors.

The system may also include a plurality of filters 36. In certainembodiments, the filters may be within a chamber of the system 28.Different chambers within the system may be comprised of differentfilters. The filters are effective to separate the regenerative cells,e.g., stem cells and/or progenitor cells, from undesirable cells anddisaggregation agents that may be used in accordance with the system. Inone embodiment, a filter assembly 36 includes a hollow fiber filtrationdevice. In another embodiment, a filter assembly 36 includes apercolative filtration device, which may or may not be used with asedimentation process. In a further embodiment, the filter assembly 36comprises a centrifugation device, which may or may not be used with anelutriation device and process. In yet another embodiment, the systemcomprises a combination of these filtering devices. The filtrationfunctions of the present invention can be two-fold, with some filtersremoving things from the final concentration such as collagen, freelipid, free adipocytes and residual collagenase, and with other filtersbeing used to concentrate the final product. The filters of the systemmay be comprised of a plurality of pores ranging in diameters and/orlength from 20 to 800 μm. In a preferred embodiment, the collectionchamber 20 has a prefixed filter 28 with a plurality of pores rangingfrom 80 to 400 μm. In another preferred embodiment, the collectionchamber 20 has a prefixed filter 28 with a plurality of 265 μm pores. Inother embodiments, the filters may be detachable and/or disposable.

The system may also be comprised of one or more temperature controldevices (not shown) that are positioned to adjust the temperature of thematerial contained within one or more chambers of the system. Thetemperature control device may be a heater, a cooler or both, i.e., itmay be able to switch between a heater and a cooler. The temperaturedevice may adjust the temperature of any of the material passing throughthe system, including the tissue, the disaggregation agents, theresuspension agents, the rinsing agents, the washing agents or theadditives. For example, heating of adipose tissue facilitatesdisaggregation whereas the cooling of the regenerative cell output isdesirable to maintain viability. Also, if pre-warmed reagents are neededfor optimal tissue processing, the role of the temperature device wouldbe to maintain the pre-determined temperature rather than to increase ordecrease the temperature.

To maintain a closed, sterile fluid/tissue pathway, all ports and valvesmay comprise a closure that maintains the sealed configuration of thesystem. The closure may be a membrane that is impermeable to fluid, airand other contaminants or it may be any other suitable closure known inthe art. Furthermore, all ports of the system may be designed such thatthey can accommodate syringes, needles or other devices for withdrawingthe materials in the chambers without compromising the sterility of thesystem.

As set forth herein, tissue may be extracted from a patient via any artrecognized method. The aspirated tissue may be extracted prior to beingplaced in the system for processing. The aspirated tissue is typicallytransferred to the collection chamber 20 through conduits 12 via asealed entry port, such as a rubber septum closed syringe needle accessport (not shown on collection chamber). Alternatively, the tissueextraction step may be part of the system. For example, the collectionchamber 20 may be comprised of a vacuum line 11 which facilitates tissueremoval using a standard cannula inserted into the patient. Thus, inthis embodiment, the entire system is attached to the patient. Thetissue may be introduced into the collection chamber 20 through an inletport 21 via a conduit such as 12 a which are part of a closed sterilepathway. The collection chamber 20 may be comprised of a plurality offlexible or rigid canisters or cylinders or combinations thereof. Forexample, the collection chamber 20 may be comprised of one or more rigidcanisters of varying sizes. The collection chamber 20 may also becomprised of one or more flexible bags. In such systems, the bag ispreferably provided with a support, such as in internal or externalframe, that helps reduce the likelihood that the bag will collapse uponthe application of suction to the bag. The collection chamber 20 issized to hold the requisite amount of saline to appropriately wash anddisaggregate the tissue prior to the wash and concentrate stage of theprocess performed in the processing chamber 30. Preferably, the volumeof tissue or fluid present in the collection chamber 20 is easilyascertainable to the naked eye. For example, to obtain regenerativecells from adipose tissue, a suitable collection chamber has thecapacity to hold 800 ml of lipoaspirate and 1200 ml of saline.Accordingly, in one embodiment, the collection chamber 20 has a capacityof at least 2 liters. In another embodiment, to separate and concentratered blood cells from blood, the collection chamber 20 has a capacity ofat least 1.5 liters. Generally, the size of the collection chamber 20will vary depending on the type and amount of tissue collected from thepatient. The collection chamber 20 may be sized to hold as little asabout 5 ml to up to about 2 liters of tissue. For smaller tissuevolumes, e.g., 5 mls to 100 mls, the tissue may be gathered in a syringeprior to transfer to the collection chamber 20.

The collection chamber 20 may be constructed using any suitablebiocompatible material that can be sterilized. In a preferredembodiment, the collection chamber 20 is constructed of disposablematerial that meets biocompatibility requirements for intravascularcontact as described in the ISO 10993 standard. For example,polycarbonate acrylic or ABS may be used. The fluid path of thecollection chamber 20 is preferably pyrogen free, i.e., suitable forblood use without danger of disease transmittal. In one embodiment, thecollection chamber 20 is constructed of a material that allows the userto visually determine the approximate volume of tissue present in thechamber. In other embodiments, the volume of tissue and/or fluid in thecollection chamber 20 is determined by automated sensors 29. Thecollection chamber 20 is preferably designed such that in an automatedembodiment, the system can determine the volume of tissue and/or fluidwithin the chamber with a reasonable degree of accuracy. In a preferredembodiment, the system senses the volume within the collection chamberwith an accuracy of plus or minus fifteen percent.

In a particular embodiment provided by way of example only, thecollection chamber 20 is in the form of a rigid chamber, for example, achamber constructed of a medical grade polycarbonate containing aroughly conical prefixed filter 28 of medical grade polyester with amesh size of 265 μm (see FIG. 5). The rigid tissue collection containermay have a size of approximately eight inches high and approximatelyfive inches in diameter; the wall thickness may be about 0.125 inches.The interior of the cylinder may be accessed through, for example, oneor more ports for suction tubing, one or more ports with tubing forconnection through sterile docking technology, and/or one or more portsfor needle puncture access through a rubber septum. The prefixed filter28 in the interior of the collection chamber 20 is preferably structuredto retain adipose tissue and to pass non-adipose tissue as, for example,the tissues are removed from the patient. More specifically, the filter28 may allow passage of free lipid, blood, and saline, while retainingfragments of adipose tissue during, or in another embodiment after, theinitial harvesting of the adipose tissue. In that regard, the filter 28includes a plurality of pores, of either the same or different sizes,but ranging in size from about 20 μm to 5 mm. In a preferred embodiment,the filter 28 includes a plurality of 400 μm pores. In a preferredembodiment, the filter 28 is a medical grade polyester mesh of around200 μm thickness with a pore size of around 265 μm and around 47% openarea. This material holds the tissue during rinsing but allows cells topass out through the mesh following tissue disaggregation. Thus, whenthe tissues are aspirated from the patient, non-adipose tissue may beseparated from adipose tissue. The same functionality could be achievedwith different materials, mesh size, and the number and type of ports.For example, mesh pore sizes smaller than 100 μm or as large as severalthousand microns would achieve the same purpose of allowing passage ofsaline and blood cells while retaining adipose tissue aggregates andfragments. Similarly, the same purpose could be achieved by use of analternative rigid plastic material, or by many other modifications thatwould be known to those skilled in the art

The system 10 may also be comprised of one or more solution sources 22.The solution source may comprise a washing solution source 23, and atissue disaggregation agent source 24, such as collagenase. Thecollection chamber 20 is comprised of closed fluid pathways that allowsfor the washing and disaggregating solutions or agents to be added tothe tissue in an aseptic manner.

The containers for the washing solution 23 and the disaggregation agents24 may be any suitable container that can hold their contents in asterile manner, e.g., a collapsible bag, such as an IV bag used inclinical settings. These containers may have conduits 12, such asconduit 12 e, coupled to the collection chamber 20 so that the washingsolution and the disaggregation agent may be delivered to the interiorof the collection chamber 20. The washing solution and thedisaggregation agent may be delivered to the interior of the collectionchamber 20 through any art-recognized manner, including simple gravitypressure applied to the outside of the containers for the saline 23and/or the disaggregation agents 24 or by placement of a positivedisplacement pump on the conduits, e.g., conduit 12 d in FIG. 4. Inautomated embodiments, the processing device of the system calculatesvarious parameters, e.g., the volume of saline and time or number ofcycles required for washing as well as the concentration or amount ofdisaggregation agent and the time required for disaggregation based oninformation initially entered by the user (e.g., volume of tissue beingprocessed). Alternatively, the amounts, times etc. can be manuallymanipulated by the user.

The tissue and/or fluid within the collection chamber should bemaintained at a temperature ranging from 30 degrees Celsius to 40degrees Celsius. In a preferred embodiment, the temperature of thesuspension inside the collection chamber is maintained at 37 degreesCelsius. In certain embodiments, if the surgical procedure ortherapeutic application needs to be delayed, the selected tissue may bestored in the collection chamber for later use. The tissue may be storedat or about room temperature or at about 4 degrees Celsius for up to 96hours.

The washing solution may be any solution known to one of skill in theart, including saline or any other buffered or unbuffered electrolytesolution. The types of tissue being processed will dictate the types orcombinations of washing solutions used. Typically, the washing solution,such as saline, enters the collection chamber 20 after the adiposetissue has been removed from the patient and placed in the collectionchamber. However, the washing solution may be delivered to thecollection chamber 20 before the adipose tissue is extracted, or may bedelivered to the collection chamber 20 concurrently with the adiposetissue. In the collection chamber 20, the washing solution and theextracted adipose tissue may be mixed by any means including the methodsdescribed below.

For example, the tissue may be washed by agitation (which maximizes cellviability and minimizes the amount of free lipid released). In oneembodiment, the tissue is agitated by rotating the entire collectionchamber 20 through an arc of varying degrees (e.g., through an arc ofabout 45 degrees to about 90 degrees) at varying speeds, e.g., about 30revolutions per minute. In other embodiments, the tissue is agitated byrotating the entire collection chamber 20, wherein the collectionchamber 20 is comprised of one or more paddles or protrusions rigidlyattached to an inside surface of the collection chamber, through an arcof varying degrees (e.g., through an arc of about 45 degrees to about 90degrees) at varying speeds, e.g., about 30 revolutions per minute. Therotation of the collection chamber 20 described above may beaccomplished by a drive mechanism attached to or in proximity with thecollection chamber 20. The drive mechanism may be a simple belt or gearor other drive mechanism known in the art. The speed of the rotation maybe, for example, 30 revolutions per minute. Generally, higher speedshave been found to generate larger volumes of free lipids and may not beoptimal.

In other embodiments, the tissue is agitated by placing a rotatableshaft 25 inside the collection chamber 20, wherein the rotatable shaftis comprised of one or more paddles 25 a or protrusions rigidly attachedto the rotatable shaft 25 which pass through the mixture as the shaft isbeing rotated. In certain embodiments, the rotatable shaft 25 withrigidly attached 25 a paddles may be rested on the bottom of thecollection chamber 20. This may be accomplished, for example, by placingthe paddle-like device into a spinning magnetic field (e.g., magneticstirrer). Alternatively, agitating of the tissue may be accomplishedusing a simple agitator known in the art, i.e. a device implementingshaking up and down without rotation. The tissue may also be washedusing any other art-recognized means including rocking, stirring,inversion, etc.

After a desired amount of wash cycles, a tissue disaggregation agent maybe delivered to the collection chamber 20 to separate the regenerativecells from the remaining adipose tissue components. The disaggregationagent may be any disaggregation agent known to one of skill in the art.Disaggregation agents that may be used include neutral proteases,collagenase, trypsin, lipase, hyaluronidase, deoxyribonuclease, membersof the Blendzyme enzyme mixture family, e.g., Liberase H1, pepsin,ultrasonic or other physical energy, lasers, microwaves, othermechanical devices and/or combinations thereof. A preferreddisaggregation agent of the invention is collagenase. The disaggregationagents may be added with other solutions. For example, saline, such assaline delivered from a saline source 23 as described above, may beadded to the adipose tissue along with or immediately followed byaddition of collagenase. In one embodiment, the washed adipose tissue ismixed with a collagenase-containing enzyme solution at or around 37° C.for about 20-60 minutes. In other embodiments, a higher concentration ofcollagenase or similar agent may be added to decrease the digestiontime. The washed adipose tissue and the tissue disaggregation agent maythen be agitated in manners similar to the agitation methods describedabove, until the washed adipose tissue is disaggregated. For example,the washed adipose tissue and the tissue disaggregation agent may beagitated by rotating the entire collection chamber through an arc ofapproximately 90 degrees, by having a shaft which contains one or morepaddles which pass through the solution as the shaft is being rotated,and/or by rotating the entire collection chamber which contains paddlesor protrusions on the inside surface of the collection chamber.

Depending on the purpose for which the adipose derived cells will beused, the adipose tissue may either be partially disaggregated, orcompletely disaggregated. For example, in embodiments in which theadipose derived cells are to be combined with a unit of adipose tissue,it may be desirable to partially disaggregate the harvested adiposetissue, to remove a portion of the partially disaggregated adiposetissue, and then continue disaggregating the remaining portion ofadipose tissue remaining in the collection chamber. Alternatively, aportion of washed adipose tissue may be removed and set aside in asample container prior to any digestion. In another embodiment,harvested adipose tissue is partially disaggregated to concentrate cellsbefore being reintroduced back into the patient. In one embodiment, theadipose tissue is mixed with a tissue disaggregation agent for a periodof time generally less than about 20 minutes. A portion of the partiallydisaggregated tissue may then be removed from the collection chamber,and the remaining partially disaggregated tissue may be furtherdisaggregated by mixing the adipose tissue with a tissue disaggregationagent for another 40 minutes. When the adipose derived cells are to beused as an essentially pure population of regenerative cells, theadipose tissue may be fully disaggregated.

After digestion, the tissue and disaggregation agent solution is allowedto settle for a period of time sufficient to allow the buoyant andnon-buoyant components of the solution to differentiate within thecollection chamber. Typically, the time ranges from about 15 seconds toseveral minutes but other times may be implemented in modifiedembodiments. The buoyant layer is comprised of the regenerative cellsthat require further washing and concentrating. The non-buoyant layercomprises blood, collagen, lipids and other non-regenerative cellcomponents of the tissue. The non-buoyant layer must be removed to thewaste chamber.

Accordingly, the collection chamber 20 is preferably comprised of anoutlet port 22 at the lowest point of the chamber such that blood andother non-buoyant components of the tissue may be drained to one or morewaste containers 40 via one or more conduits 12. The collection chamber20 is generally in (or may be placed in) an upright position such thatthe outlet ports 22 are located at the bottom of the collection chamber.The draining may be passive or active. For example, the non-buoyantcomponents described above could be drained using gravity, by applyingpositive or negative pressure, by use of pumps 34 or by use of vents 32.In automated embodiments, the processing device can signal certainvalves and/or pumps to drain the non-buoyant layer from the collectionchamber 20. The automated embodiments may also be comprised of sensors29 which can detect when the interface between the buoyant andnon-buoyant liquids has been reached. The automated embodiments may alsobe comprised of a sensor 29, e.g., an optical sensor, which may becapable of detecting a change in the light refraction of the effluentwhich is flowing in the conduit leading out of the collection chamber.The appropriate change in the light refraction may signal the presenceof the buoyant layer in the outgoing conduits which indicates that thenon-buoyant layer has been drained. The sensor 29 can then signal theprocessing device to proceed with the next step.

In certain embodiments however, the tissue may be processed to retrievethe non-regenerative cell component of the tissue. For example, incertain therapeutic or research applications, collagen, proteins, matrixor stromal components, lipids, adipocytes or other components of thetissue may be desired. In such embodiments, it is the buoyant layercomprising the regenerative cells that must be removed as describedabove to the waste chamber. The non-buoyant layer is then retained inthe system for further processing as needed.

Once the non-buoyant layer is removed, the buoyant layer comprising theregenerative cells may be washed one or more times to remove residualcontaminants. Accordingly, the collection chamber 20 typically includesone or more ports 21 for permitting the washing solution to be deliveredto the interior of the chamber, and one or more ports 22 for permittingwaste and other materials to be directed out from the collection chamber20. For example, the collection chamber may include one or more sealedentry ports as described herein. The collection chamber 20 may alsoinclude one or more caps (not shown), such as a top cap and a bottom capto further ensure that the system remains sterile while washing solutionis delivered into the collection chamber and/or waste is transportedout. The ports 21 may be provided on the caps of the collection chamberor on a sidewall of the collection chamber.

The process of washing with fresh wash solution may be repeated untilthe residual content of non-buoyant contaminants in the solution reachesa pre-determined level. In other words, the remaining material in thecollection chamber 20, which comprises the buoyant material of themixture described above, including adipose tissue fragments, may bewashed one or more additional times until the amount of undesiredmaterial is reduced to a desired pre-determined level. One method ofdetermining the end point of the washing is to measure the amount of redblood cells in the tissue solution. This can be accomplished bymeasuring the light absorbed on the 540 nm wavelength. In a preferredembodiment, a range between about 0.546 and about 0.842 is deemedacceptable.

During the washing and/or disaggregation, one or more additives may beadded to the various containers as needed to enhance the results. Someexamples of additives include agents that optimize washing anddisaggregation, additives that enhance the viability of the active cellpopulation during processing, anti-microbial agents (e.g., antibiotics),additives that lyse adipocytes and/or red blood cells, or additives thatenrich for cell populations of interest (by differential adherence tosolid phase moieties or to otherwise promote the substantial reductionor enrichment of cell populations). Other possible additives includethose that promote recovery and viability of regenerative cells (forexample, caspase inhibitors) or which reduce the likelihood of adversereaction on infusion or emplacement (for example, inhibitors ofre-aggregation of cells or connective tissue).

After a sufficient settling time has elapsed, the non-buoyant fractionof the resulting mixture of washed adipose tissue fragments and tissuedisaggregation agents will contain regenerative cells, e.g., stem cellsand other adipose derived progenitor cells. As discussed herein, thenon-buoyant fraction containing the regenerative cells will betransferred to the processing chamber 30 wherein the regenerative cellsof interest, such as the adipose derived stem cells, will be separatedfrom other cells and materials present in the non-buoyant fraction ofthe mixture. This non-buoyant fraction is referred to herein as theregenerative cell composition and comprises multiple different types ofcells, including stem cells, progenitor cells, endothelial precursorcells, adipocytes and other regenerative cells described herein. Theregenerative cell composition may also contain one or more contaminants,such as collagen and other connective tissue proteins and fragmentsthereof, which were present in the adipose tissue fragments, or residualcollagenase from the tissue disaggregation process.

The processing chamber 30 of the invention is preferably positionedwithin the system such that the regenerative cell composition moves fromthe collection chamber 20 to the processing chamber 30 by way of tubing12, valves 14 and pump 34 in a sterile manner. The processing chamber issized to accommodate tissue/fluid mixtures ranging from 10 mL to 1.2 L.In a preferred embodiment, the processing chamber is sized toaccommodate 800 mLs. In certain embodiments, the entire regenerativecell composition from the collection chamber 20 is directed to theprocessing chamber 30. However, in other embodiments, a portion of theregenerative cell composition is directed to the processing chamber 30,and another portion is directed to a different region of the system,e.g., the sample chamber 60, to be recombined with cells processed inthe processing chamber 30 at a later time.

The processing chamber 30 may be constructed using any suitablebiocompatible material that can be sterilized. In a preferredembodiment, the processing chamber 30 is constructed of disposablematerial that meets biocompatibility requirements for intravascularcontact, as described in the ISO 10993 standard. For example,polycarbonate, acrylic, ABS, ethylene vinyl acetate or styrene-butadienecopolymers (SBC) may be used. In another embodiment, the fluid path ofthe disposable processing chamber is pyrogen free. The processingchamber may be in the form of a plastic bag, such as thoseconventionally used in processing blood in blood banks; or in otherembodiments, it may be structurally rigid (FIG. 6). In one embodiment,the processing chamber 30 may be similar to the processing chamberdisclosed in commonly owned U.S. application Ser. No. 10/316,127, filedDec. 7, 2001 and U.S. application Ser. No. 10/325,728, filed Dec. 20,2002, the contents of which in their entirety are hereby incorporated byreference.

The processing chamber 30 may be constructed in any manner suitable forseparating and concentrating cells, including filtration andcentrifugation and/or combinations thereof. In certain embodiments, theregenerative cell composition from the collection chamber 20 isintroduced into the processing chamber 30 where the composition can befiltered to separate and/or concentrate a particular regenerative cellpopulation. Cell filtration is a method of separating particularcomponents and cells from other different components or types of cells.For example, the regenerative cell composition of the inventioncomprises multiple different types of cells, including stem cells,progenitor cells and adipocytes, as well as one or more contaminants,such as collagen, which was present in the adipose tissue fragments, orresidual collagenase from the tissue disaggregation process. The filters36 present in the processing chamber 30 may allow for separation andconcentration of a particular subpopulation of regenerative cells, e.g.,stem cells or endothelial progenitors cells etc.

Some variables which are associated with filtration of cells from aliquid include, but are not limited to, pore size of the filter media,geometry (shape) of the pore, surface area of the filter, flow directionof the solution being filtered, trans-membrane pressure, dilution of theparticular cell population, particulate size and shape as well as cellsize and cell viability. In accordance with the disclosure herein, theparticular cells that are desired to be separated or filtered aretypically adipose derived stem cells. However, in certain embodiments,the particular cells may include adipose derived progenitor cells, suchas endothelial precursor cells, alone or in combination with the stemcells.

The regenerative cell composition may be directed through a filterassembly, such as filter assembly 36. In certain embodiments, the filterassembly 36 comprises a plurality of filters which are structured toperform different functions and separate the regenerative cellcomposition into distinct parts or components. For example, one of thefilters may be configured to separate collagen from the regenerativecell composition, one of the filters may be configured to separateadipocytes and/or lipid components from the regenerative cellcomposition, and one of the filters may be configured to separateresidual enzymes, such as the tissue disaggregation agent, from theregenerative cell composition. In certain embodiments, one of thefilters is capable of performing two functions, such as separatingcollagen and the tissue disaggregation agent from the composition. Theplurality of filters are typically serially arranged; however, at leasta portion of the filters may be arranged in parallel, as well. A serialarrangement of the filters of the filter assembly 36 is shown in FIG. 2.A parallel arrangement of the filters of the filter assembly 36 is shownin FIG. 3.

In one embodiment, the filter assembly 36 comprises a first filter, asecond filter, and a third filter. The first filter is configured toremove collagen particles present in the regenerative cell composition.These collagen particles are typically approximately 0.1 microns indiameter and can be up to 20 microns long. The collagen particles may beof varying sizes depending on the digestion. They also may be fibrils,meaning they have twists and turns. Any of the filters described hereinmay be made from polyethersulfone, polyester, PTFE, polypropylene, PVDF,or possibly cellulose. There are two possibilities for filtering thecollagen. One is to try to remove the larger particles first, lettingthe cells go through, which would require for example a filter probablyin the 10 micron range. The second method is to use a smaller sizefilter, such as 4.5 micron, with the intent that the collagen would bewell digested, so as to trap the cells, and let the collagen passthrough. This would require a means to float the cells back off thefilter. There may also be a possibility of implementing a filter whichwould attract and hold the collagen fibers.

The second filter is configured to remove free immature adipocytes whichare not buoyant in the regenerative cell composition. In one embodimentthe second filter can be constructed of polyester and have a pore sizebetween about 30 and about 50 microns with a preferred pore size beingabout 40 microns. Although referred to as a second filter, placement ofsuch a device may be in a first, rather than second, position tofacilitate an initial removal of larger cells and particles. The thirdfilter is configured to remove the unused or residual collagenase orother tissue disaggregation agent present in the composition. In apreferred implementation, the collagenase may degenerate over time. Inone embodiment, the third filter comprises a plurality of pores having adiameter, or length less than 1 μm. In certain embodiments, the poresmay have diameters that are smaller than 1 μm. In other embodiments, thepores have diameters between 10 kD and 5 microns. In certainembodiments, the third filter may be configured to concentrate theregenerative cell population into a small volume of saline or otherwashing solution, as discussed herein. As presently preferred, only thefinal filter is the hollow fiber unit. It is not necessary for any ofthe filters to be of the hollow fiber type. The hollow fiber unit isused for the final filter in a preferred implementation because it isthe most efficient in removing the collagenase with the smallestdetrimental effect to the regenerative cells. In an embodiment whereinthe device is a collection of off the shelf items, the three filters arein separate housings. It is feasible to have the first and secondfilters combined into one housing if a hollow fiber unit is used for thethird filter. If the final filter is not a hollow fiber set-up then allthree filters can be contained in one housing.

The filters of the filter assembly 36 may be located in the processingchamber 30 or may be provided as components separate from the processingchamber 30. In addition, the filters of the filter assembly 36 may beprovided in multiple processing chambers or in an inline fashion. Incertain embodiments, the conduits or tubing may act as a processingchamber or chambers. The processing chamber can be reduced in size suchthat it becomes the inside volume of the conduits which connect thefilters. This type of system will function correctly if the volume oftissue solution is sized appropriately. Thus, the conduits may act asthe processing chamber by containing the fluid with cells as it is beingrun through the filters. Care may be taken to minimize the volume of theconduits so that cells/tissue are not unnecessarily lost in the processof priming and running the system.

Referring to the embodiment described above, the regenerative cellcomposition, containing the washed cells and residual collagen,adipocytes, and/or undigested tissue disaggregation agent, may bedirected through the first filter to remove at least a portion of andpreferably substantially all of the collagen particles from thecomposition so that fewer, and preferably no, collagen particles arepresent in the filtered solution. The filtered regenerative cellcomposition containing the adipocytes and/or undigested tissuedisaggregation agent, may then be directed through the second filter toremove at least a portion of and preferably substantially all of thefree adipocytes from the filtered regenerative cell composition.Subsequently, the twice filtered regenerative cell composition,containing the undigested tissue disaggregation agent, may be directedthrough the third filter, such as a hollow fiber filtration device, asdiscussed herein, to remove or reduce the undigested tissuedisaggregation agent from the regenerative cell composition.

The thrice-filtered regenerative cell composition (i.e., the compositionremaining after being passed through the first, second, and thirdfilters) may then be directed to multiple outlets, which may include aportion of the processing chamber 30 comprising multiple outlets. Theseoutlets can serve to maintain the necessary pressure, as well as toprovide connections via conduits to other containers which may includethe collection chamber 20, the output chamber 50, and/or the wastecontainer 40.

In one embodiment, a filter of the filter assembly 36 comprises ahollow-fiber filtration member. Or, in other words, the filter comprisesa collection of hollow tubes formed with the filter media. Examples offilter media which can be used with the disclosed system 10 includepolysulfone, polyethersulfone or a mixed ester material, and the like.These hollow fibers or hollow tubes of filter media may be contained ina cylindrical cartridge of the filter assembly 36. The individual tubesor fibers of filter media typically have an inside diameter which rangesfrom about 0.1 mm to about 1 mm with a preferred value being about 0.5mm. The diameter and length of a suitable cylindrical cartridge willdetermine the number of individual tubes of filter media which can beplaced inside the cartridge. One example of a suitable hollow fiberfilter cartridge is the FiberFlo® Tangential Flow Filter, catalog#M-C-050-K(Minntech, Minneapolis, Minn.). Pore sizes of the filter mediacan range between about 10 kiloDaltons and about 5 microns with apreferred pore size being about 0.5 microns.

In the hollow-fiber filter, each hollow tube has a body with a firstend, a second end, and a lumen located in the body and extending betweenthe first end and second end. The body of each hollow tube includes aplurality of pores. The pores are generally oriented in the body so thata regenerative cell composition is filtered by flowing through the lumenof the body, and the products to be filtered tangentially pass throughthe pores, as shown in FIG. 12A. In other words, the smaller particlesin the liquid pass tangentially through the pores relative the flow offluid through the lumen of the body. The composition with theregenerative cells passes through the lumen of each hollow tube when thecomposition is being filtered. Preferably, the flow of the compositionis tangential to the pores of the body of each hollow tube.

By using a tangential flow of fluid, the efficiency of filtration of thestem cells may be enhanced relative to other filtration techniques. Forexample, in accordance with some filtration techniques, the pores of thefilter media are placed in such a manner that the filter is orientatedperpendicular to the flow of the fluid so that the Filter media blocksthe path of the fluid being filtered, as illustrated in FIG. 12B. Inthis type of filtration, the particles which are being filtered out ofthe regenerative cell composition, e.g., the stem cells, tend to buildup on one side of the filter and block the flow of the fluid through thepores. This blockage can reduce the efficiency of the filter. Inaddition, the cells are constantly compressed by the pressure of thefluid flow as well as the weight of the cells accumulating on theupstream side of the filter. This can lead to increased lysis of stemcells. Thus, in such filtration techniques wherein the flow of fluid isparallel to the orientation of the pores in the filter, both large cellsand small particles can be undesirably directed against the filter mediaas the fluid is passed through the pores. Consequently, larger productsin the liquid such as cells may block the pores, thereby decreasing thefiltering effect and increasing an occurrence of cell rupture or injury.

In contrast, in the hollow fiber configuration of the present system 10,the fluid which is being filtered flows inside the lumen of the hollowtube. The portion of the fluid which has the ability to pass through thepores of the body of the filter does so with the aid of the positivepressure of the fluid on the inside of the body as well as a negativepressure which is applied on the outside of the body. In thisembodiment, the cells typically are not subjected to the pressure of thefluid flow or the weight of other cells, and therefore, the shear forceson the stem cells are reduced Thus, the efficiency and effectiveness ofthe filtration can be enhanced by the reduction in clogging rates andthe reduction in regenerative cell lysis. Due to the size of the salineand unwanted protein molecules, during filtration, these molecules andother small components pass through the pores of the bodies of thehollow tubes to the outside of the hollow tubes and are directed to thewaste container 40. In one embodiment, filtration is enhanced bygenerating a vacuum on the outside of the hollow tube filter media. Dueto the size of the regenerative cells, e.g., stem cells or progenitorcells, these cells typically cannot pass through the pores of the bodyand therefore remain on the inside of the hollow tube filter (e.g., inthe lumens of the tubes) and are directed back to the processing chamber30 via a conduit between the filter and the processing chamber, or tothe output chamber 50.

In one specific embodiment, the hollow fiber filter has about a 0.05micron pore size, and contains approximately 550 cm² surface area offilter media. An individual media tube typically has a diameter of about0.5 mm. In processing 130 ml of the regenerative cell composition,approximately 120 ml of additional saline may be added to thecomposition. The processing or filter time may be approximately 8minutes. The differential of the pressures on either side of the body ofthe hollow fiber tube (e.g., the pressure inside the lumen of the body,and outside the body) is considered the trans-membrane pressure. Thetrans-membrane pressure can range from about 1 mmHg to about 500 mmHgwith a preferred pressure being about 200 mmHg. The average nucleatedcell recovery and viability using hollow fiber filtration can beapproximately 80% of viable cells.

The amount of collagenase which is typically removed in such a systemequates to a three log reduction. For example if the initialconcentration of collagenase in the regenerative cell composition whichis transferred from the collection chamber to the processing chamber is0.078 U/ml the collagenase concentration of the final regenerative cellcomposition would be 0.00078 U/ml. The collagenase is removed in thehollow fiber filter, and the hollow fiber filter corresponds to thethird filter discussed above.

Processing chambers illustrating one or more cell filtration methodsdescribed above are shown in the Figures, particularly FIGS. 1-3. Withreference to FIGS. 1-3, between the processing chamber 30 and thefiltering chamber of the filter assembly 36, a pump may be provided,such as pump 34. In addition, vent and pressure sensors, such as vent32, and pressure sensor 39, may be provided in line with the processingchamber 30 and the filter assembly 36. Fittings for the output chamber50 may also be provided. These optional components (e.g., the pump 34,the vent 32, the pressure sensor 39, and the fittings for the outputchamber 50) may be provided between the processing chamber 30 and thefilter assembly 36 so that liquid contained in the processing chamber 30may flow to one or more of these optional components before flowingthrough the filter assembly 36. For example, liquid may flow through thepump 34 before it is passed to the filter assembly 36. Or, liquid maypass through the pressure sensor 39 before passing through the filterassembly to obtain a pre-filter liquid pressure in the system. Incertain situations, one or more of these components may also be providedas an element of the processing chamber 30, such as the vent 32 asillustrated in FIG. 6. In the illustrated embodiment, the pressuresensor 39 is in line to determine the pressure of the regenerative cellcomposition which is generated by the pump 34 as it enters the filteringchamber of the filter assembly 36. This construction can facilitatemonitoring of the trans-membrane pressure across the filter membrane.Additional saline or other buffer and washing solution can be added tothe regenerative cell composition to assist in the removal of unwantedproteins as the composition is being filtered through the filterassembly 36. This repeated washing can be performed multiple times toenhance the purity of the regenerative cells. In certain embodiments,the saline can be added at any step as deemed necessary to enhancefiltration.

In one specific embodiment, which is provided by way of example and notlimitation, the unwanted proteins and saline or other washing solutionis removed in the following manner. The composition with theregenerative cells, as well as collagen and connective tissue particlesor fragments, adipocytes, and collagenase, is cycled through a series offilters until a minimum volume is reached. The minimum volume is afunction of the total hold up volume of the system and somepredetermined constant. The hold up volume is the volume of liquid whichis contained in the tubing and conduits if all of the processingchambers are empty. In one embodiment, the minimum volume is 15 ml. Whenthe minimum volume is reached, a predetermined volume of washingsolution is introduced into the system to be mixed with the regenerativecell composition. This mixture of washing solution and the regenerativecell composition is then cycled through the filters until the minimumvolume is reached again. This cycle can be repeated multiple times toenhance the purity of the regenerative cells, or in other words, toincrease the ratio of regenerative cells in the composition to the othermaterials in the composition. See FIGS. 10 and 11.

After it has been determined that the regenerative cell composition hasbeen cleansed of unwanted proteins and concentrated sufficiently (inexemplary embodiments, minimum concentrations within a range of about1×10⁵ to about 1×10⁷ cells/m¹ can be used and, in a preferred embodimentthe minimum concentration can be about 1×10⁷ cells/ml), an outputchamber 50, such as an output bag, may be connected to an outlet port ofthe processing chamber 30 and/or the filter assembly 36, depending onthe specific embodiment. A vent, such as the vent 32, may then be openedto facilitate the output of the concentrated regenerative cells. In oneimplementation, this determination of when a minimum concentration hasbeen reached is made empirically after experiments have been run andprogrammed into the electronic controls of the device. The determinationcan be an input into the process of what is desired to yield, i.e., howmany stem/progenitor cells are desired, or range of cell concentration.Based on scientific data, a predefined amount of adipose tissue needs tobe obtained and placed into the system to achieve the desired output.With the vent 32 open, a pump, such as the pump 34, can function totransfer the concentrated regenerative cells into the output bag. In oneembodiment, the output bag 50 is similar to an empty blood bag which hasa tube with a fitting on one end. In a sterile fashion, the fitting onthe output bag may be attached to the outlet port, and the concentratedregenerative cells may be transferred to the output bag.

As illustrated in FIGS. 1-3, a vacuum pump 26 may be provided in thesystem 10 to change the pressure in the system, among other things. Forexample, the vacuum pump 26 may be coupled to the collection chamber 20via a conduit, such as conduit 12 b, to cause a decrease in pressurewithin the collection chamber 20. Vacuum pump 26 may also be coupled tothe processing chamber 30 by way of a conduit, such as conduit 12 g.Regarding the operation of vacuum pump 26 in connection with pump 34,two separate vacuum pumps or sources may be implemented, or a single onemay be implemented by using valves which direct the vacuum pull to thedifferent conduits that need it at specific points in the process. Inaddition, vacuum pump 26 may be coupled to the waste container 40 via aconduit, such as conduit 12 f.

With reference to FIGS. 10 and 11, the pressure generated by the vacuumpump 26 can be used to direct the flow of fluids, including theregenerative cells, through the conduits 12. This pressure can besupplied in multiple directions, for example, by automatically ormanually controlling the position of one or more valves 14 in the system10. The system 10 can be made to function properly with the use ofpositive pressure or through the use of negative pressure, orcombinations thereof. For instance, the regenerative cells can be pulledthrough the first and second filters described above into a soft sidedcontainer which is connected to the third filter. The soft-sidedcontainer can be in line (serial) connected ahead of the third filter.The final output chamber may be a soft sided container which is on theother side (e.g., the downstream side) of the third filter. In thisembodiment, pressure is used to move the regenerative cells from onesoft sided container to a second soft sided container through thefilter.

In another embodiment of the system 10, the filtration of the stem cellsand/or adipose derived progenitor cells may be accomplished using acombination of percolative filtration and sedimentation. For example,such a system uses saline that is passed through a tissue regenerativecell composition (e.g., the composition containing the stem cells and/oradipose derived progenitor cells) and then through a filter. Some of thevariables which are associated with percolative filtration of cells froma regenerative cell composition include, but are not limited to, poresize of the filter media, pore geometry or shape, surface area of thefilter, flow direction of the regenerative cell composition beingfiltered, flow rate of the infused saline, trans-membrane pressure,dilution of the cell population, cell size and viability.

In one embodiment of the system 10, the processing chamber 30 uses afilter assembly 36 which implements percolative filtration andsedimentation to separate and concentrate the regenerative cells. By wayof example, and not by way of limitation, the processing chamber 30 isdefined as a generally cylindrical body having a sidewall 30 a, a topsurface 30 b, and a bottom surface 30 c, as shown in FIG. 6. A sterilevent 32 is provided in the top surface 30 b.

In the embodiment of FIG. 6, the processing chamber 30 is illustrated asincluding a filter assembly 36, which includes two filters, such aslarge pore filter 36 a, and small pore filter 36 b. The pore sizes ofthe filters 36 a and 36 b typically are in a range between about 0.05microns and about 10 microns. The large pore filter 36 a may comprisepores with a diameter of about 5 μm, and the small pore filter 36 b maycomprise pores with a diameter of about 1-3 μm. In one embodiment, thefilters have a surface area of about 785 mm². Filters 36 a and 36 bdivide an interior of the processing chamber 30 to include a firstchamber 37 a, a second chamber 37 b, and a third chamber 37 c. As shownin FIG. 6, first chamber 37 a is located between second chamber 37 b andthird chamber 37 c. In addition, first chamber 37 a is shown as beingthe region of the processing chamber 30 having an inlet port 31 a and anoutlet port 31 b. The illustrated processing chamber 30 includes aplurality of ports providing communication paths from an exterior of theprocessing chamber 30 to the interior of the processing chamber 30, suchas ports 31 a, 31 b, and 31 c. The ports 31 a, 31 b, and 31 c, areillustrated as being disposed in the sidewall 30 a of a body of theprocessing chamber 30. However, the ports 31 a, 31 b, and 31 c could bepositioned in other regions, as well. Port 31 a is illustrated as asample inlet port, which is constructed to be coupled to a conduit sothat a composition containing regenerative cells can be passed into theinterior of the processing chamber 30. Port 31 b is illustrated as anoutlet port constructed to be coupled to a conduit so that the separatedand concentrated cells may be removed from the interior of theprocessing chamber 30. Port 31 c is illustrated as an inlet portconstructed to be coupled to a conduit for delivery of a fresh washingsolution, such as saline into the interior of the processing chamber 30.

In use, the regenerative cells may be introduced into the centralchamber 37 a via inlet port 31 a. Saline or other buffer is introducedinto the bottom chamber 37 b through inlet port 31 c. The saline may bedirected through the regenerative cell composition in chamber 37 a at arate of about 10 ml/min. The flow rate of the saline is such that itcounteracts the force of gravity. The flow of saline gives the cells inthe chamber the ability to separate based on the density of the cells.Typically, as the saline is forced up through the composition the largercells in the composition will settle to the bottom of the centralchamber 37 a, and the smaller cells and proteins will be carried awaythrough the second filter 36 b into the top chamber 37 c. This filteringis accomplished by adjusting the flow rate of the saline such that thelarger cells are rolled in place which allows the smaller particles tobe liberated and carried off with the saline. The sterile vent 32 isincluded in the chamber 30 to ensure that the correct pressure gradientis maintained in the three chambers within the processing unit. Theupper chamber 37 c can comprise an absorbent media 33. The purpose ofthe absorbent media is to trap the unwanted proteins in the solution toensure that they do not cross the filter media back into the processingsolution, if, for example, the saline flow rate decreases. An absorbentmedia can be a type of filter material that is absorbent, or attractsmaterials or components to be filtered out. An outflow port can be addedabove the top filter to help draw off the waste. Another embodiment ofthis may be to apply a gentle vacuum from the top to help pull offwaste. Absorbent media can be implemented when, as in the illustratedembodiment, the flow rates are relatively small. Excess saline andproteins are then carried away to a waste container.

When the larger cells, (e.g., the adipose derived stem cells and/orprogenitor cells) have been sufficiently separated from smaller cellsand proteins, the composition containing the separated cells may beconcentrated, as discussed herein. The composition may be furtherconcentrated after it has been removed from chamber 37 a through outletport 31 b, or while it is in the chamber 37 a. In one embodiment, theconcentration of cells in the composition is increased in the followingmanner. After the cells have been sufficiently separated the filters,such as filters 36 a and 36 b, may be moved towards each other. Thismovement has the effect of reducing the volume between the two filters(e.g., the volume of chamber 37 a). A vibrating member may also beprovided in connection with the processing chamber 30 to facilitateconcentrating of the cells in the composition. In one embodiment, thevibrating member may be coupled to the filter 36 b (e.g., the small porefilter). Vibrating can reduce an incidence of cells becoming trapped inthe filters. The reduction in volume of the composition allows theexcess saline to be removed as waste and the cells to be concentrated ina smaller volume.

In another embodiment, the concentration of the regenerative cells isaccomplished in the following manner. After the cells have beensufficiently separated, the regenerative cell composition can betransferred to another chamber (not shown) which uses gravity to filterout the excess saline. In a preferred embodiment, the sedimentation canoccur at the same time as the percolation. This sedimentation may beaccomplished by introducing the composition on top of a filter which hasa pore size ranging from about 10 kD to about 2 microns. In oneembodiment, a suitable filter has a pore size of about 1 micron. Theforce of gravity will allow the saline and smaller particles to bepassed through the filter while preventing the cells in the compositionto flow through the filter. After the desired concentration of cells hasbeen obtained, and after the filtered smaller particles have beenremoved from below the filter, the regenerative cell composition may beagitated to remove the cells from the filter and, subsequently, theconcentrated regenerative cells may be transferred to the output bag.The smaller particles can be drawn off as waste through an outlet.

In a particular embodiment, the regenerative cell composition from thecollection chamber 20 is transported to the processing chamber 30wherein the composition can be centrifuged to separate and concentrateregenerative cells. Centrifugation principles are well know in the artand will be not be repeated herein in the interest of brevity. Standard,art-recognized centrifugation devices, components and parameters areutilized herein. An exemplary processing chamber for use as part of acentrifuge device is shown in FIGS. 7 and 8. Typically, a centrifugedevice causes a centrifuge chamber (such as the one shown in FIG. 7) tospin around an axis to thereby increasing the force on the cells in thesolution to be greater than gravity. The denser or heavier materials inthe solution typically settle to one end of the centrifuge chamber,i.e., an output chamber 50 of FIG. 7, to form a regenerative cellpellet. The pellet may then be re-suspended to obtain a solution with adesired concentration of cells and/or a desired volume of cells andmedium. The processing chamber shown in FIG. 7 is constructed toseparate and concentrate cells using both centrifugal and gravitationalforces. Specifically, during centrifugation, centrifugal force directsthe denser components of the regenerative cell composition, e.g., theregenerative cells, towards the outermost ends of the centrifugechamber. As the centrifuge chamber slows down and eventually stops,gravitational force helps the regenerative cells to remain in theoutermost ends of the centrifuge chamber and form a cell pellet.Accordingly, the unwanted components of the regenerative cellcomposition, i.e., the waste, can be removed without disturbing the cellpellet.

In yet another embodiment of the invention, the processing chamber maybe comprised of a cell concentrator in the form of a spinning membranefilter. In a further embodiment of the centrifugation process,centrifugal elutriation may also be applied. In this embodiment, thecells may be separated based on the individual cell sedimentation ratesuch that the directional (e.g., outward) force applied bycentrifugation causes cells and solutes to sediment at different rates.In elutriation, the sedimentation rate of the target cell population isopposed by an opposite (e.g., inward) flow rate applied by pumpingsolution in the opposite direction to the centrifugal force. Thecounterflow is adjusted so that the cells and particles within thesolution are separated. Elutriation has been applied in many instancesof cell separation (Inoue, Carsten et al. 1981; Hayner, Braun et al.1984; Noga 1999) and the principles and practices used to optimize flowand centrifugal parameters can be applied herein in light of the presentdisclosure by one skilled in the art.

FIG. 9 illustrates principles associated with an elutriationimplementation in accordance with the present invention. The elutriationembodiment can be similar to a centrifugation implementation to theextent that a force is applied to the solution using a spinning rotor.Some of the variables which are associated with the presently embodiedelutriation separation include, but are not limited to, the size andshape of the spinning chamber, the diameter of the rotor, the speed ofthe rotor, the diameter of the counter flow tubing, the flow rate of thecounter flow, as well as the size and density of the particles and cellswhich are to be removed from solution. As in centrifugation, theregenerative cells can be separated based on individual cell densities.

In one embodiment the regenerative cell composition, e.g., the solutioncontaining the regenerative cells and the collagenase, is introducedinto a chamber of a spinning rotor, as shown in FIG. 9.1. After thesolution is added to the chamber additional saline is added to thechamber at a predetermined flow rate. The flow rate of the saline can bepredetermined as a function of the speed of the rotor, the celldiameter, and the chamber constant which has been establishedempirically. The flow rate will be controlled for example with a devicesimilar to an IV pump. A purpose of the additional saline is to providea condition inside the rotor chamber where the larger particles willmove to one side of the chamber and the smaller particles will move tothe other, as illustrated in FIG. 9.2. The flow is adjusted so that, inthis application, the smaller particles will exit the chamber and moveto a waste container, as shown in FIG. 9.3. This movement results in thesolution in the rotor chamber having a substantially homogenouspopulation of cells, such as stem cells. After it has been determinedthat the stem cells have been separated from the rest of the items inthe solution (with unwanted proteins and free lipids having been removedfrom the chamber), the counter flow is stopped. The cells inside thechamber will then form a concentrated pellet on the outside wall of thechamber. The counter flow is reversed and the cell pellet is transferredto the output bag.

As previously set forth herein, the processing chamber 30 or the outputchamber 50 may include one or more ports, e.g., ports 51 or 52. One ormore of these ports may be designed to transport the regenerative cellsobtained using any combination of methods described above, or a portionthereof, via conduits to other surgical devices, cell culturing devices,cell marinading devices, gene therapy devices or purification devices.These ports may also be designed to transport the regenerative cells viaconduits to additional chambers or containers within the system or aspart of another system for the same purposes described above. The portsand conduits may be also be used to add one or more additives, e.g.,growth factors, re-suspension fluids, cell culture reagents, cellexpansion reagents, cell preservation reagents or cell modificationreagents including agents that transfer genes to the cells. The portsand conduits may also be used to transport the regenerative cells toother targets such as implant materials (e.g., scaffolds or bonefragments) as well as other surgical implants and devices.

Further processing of the cells may also be initiated by reconfiguringthe interconnections of the disposable sets of the existing system,re-programming the processing device of the existing system, byproviding different or additional containers and/or chambers for theexisting system, by transporting the cells to a one or more additionalsystems or devices and/or any combinations thereof. For example, thesystem can be reconfigured by any of the means described above such thatthe regenerative cells obtained using the system may be subject to oneor more of the following: cell expansion (of one or more regenerativecell types) and cell maintenance (including cell sheet rinsing and mediachanging); sub-culturing; cell seeding; transient transfection(including seeding of transfected cells from bulk supply); harvesting(including enzymatic, non-enzymatic harvesting and harvesting bymechanical scraping); measuring cell viability; cell plating (e.g., onmicrotiter plates, including picking cells from individual wells forexpansion, expansion of cells into fresh wells); high throughputscreening; cell therapy applications; gene therapy applications; tissueengineering applications; therapeutic protein applications; viralvaccine applications; harvest of regenerative cells or supernatant forbanking or screening, measurement of cell growth, lysis, inoculation,infection or induction; generation of cells lines (including hybridomacells); culture of cells for permeability studies; cells for RNAi andviral resistance studies; cells for knock-out and transgenic animalstudies; affinity purification studies; structural biology applications;assay development and protein engineering applications.

For example, if expansion of a regenerative cell population is requiredfor a particular application, an approach using culture conditions topreferentially expand the population while other populations are eithermaintained (and thereby reduced by dilution with the growing selectedcells) or lost due to absence of required growth conditions could beused. Sekiya et al have described conditions which might be employed inthis regard for bone marrow-derived stem cells (Sekiya et al., 2002).This approach (with or without differential adherence to the tissueculture plastic) could be applied to a further embodiment of thisinvention. In this embodiment the final regenerative cell pellet isremoved from the output chamber and placed into a second systemproviding the cell culture component. This could be in the form of aconventional laboratory tissue culture incubator or a Bioreactor-styledevice such as that described by Tsao et al., U.S. Pat. No. 6,001,642,or by Armstrong et al., U.S. Pat. No. 6,238,908. In an alternativeembodiment, the cell expansion or cell culture component could be addedto the existing system, e.g., into the output chamber, allowing forshort-term adherence and/or cell culture of the adipose derived cellpopulations. This alternate embodiment would permit integration of thecell culture and/or cell expansion component to the system and removethe need for removing the cells from this system and placement withinanother.

During the processing, one or more additives may be added to or providedwith the various chambers or containers as needed to enhance theresults. These additives may also be provided as part of another systemassociated with the existing system or separate from the existingsystem. For example, in certain embodiments, the additives are added orprovided without the need for removing the regenerative cells from thesystem. In other embodiments, the additives are added or provided byconnecting a new container or chamber comprising the additives into anunused port of the system in a sterile manner. In yet other embodiments,the additives are added or provided in a second system or device that isnot connected to the system of the present invention. Some examples ofadditives include agents that optimize washing and disaggregation,additives that enhance the viability of the active cell populationduring processing, anti-microbial agents (e.g., antibiotics), additivesthat lyse adipocytes and/or red blood cells, or additives that enrichfor cell populations of interest (by differential adherence to solidphase moieties or to otherwise promote the substantial reduction orenrichment of cell populations) as described herein.

For example, to obtain a homogenous regenerative cell population, anysuitable method for separating and concentrating the particularregenerative cell type may be employed, such as the use of cell-specificantibodies that recognize and bind antigens present on, for example,stem cells or progenitor cells, e.g., endothelial precursor cells. Theseinclude both positive selection (selecting the target cells), negativeselection (selective removal of unwanted cells), or combinationsthereof. Intracellular markers such as enzymes may also be used inselection using molecules which fluoresce when acted upon by specificenzymes. In addition, a solid phase material with adhesive propertiesselected to allow for differential adherence and/or elution of aparticular population of regenerative cells within the final cell pelletcould be inserted into the output chamber of the system.

An alternate embodiment of this differential adherence approach wouldinclude use of antibodies and/or combinations of antibodies recognizingsurface molecules differentially expressed on target regenerative cellsand unwanted cells. Selection on the basis of expression of specificcell surface markers (or combinations thereof) is another commonlyapplied technique in which antibodies are attached (directly orindirectly) to a solid phase support structure (Geiselhart et al., 1996;Formanek et al., 1998; Graepler et al., 1998; Kobari et al., 2001; Mohret al., 2001).

In another embodiment the cell pellet could be re-suspended, layeredover (or under) a fluid material formed into a continuous ordiscontinuous density gradient and placed in a centrifuge for separationof cell populations on the basis of cell density. In a similarembodiment continuous flow approaches such as apheresis (Smith, 1997),and elutriation (with or without counter-current) (Lasch et al., 2000)(Ito and Shinomiya, 2001) may also be employed.

Other examples of additives may include additional biological orstructural components, such as cell differentiation factors, growthpromoters, immunosuppressive agents, medical devices, or anycombinations thereof, as discussed herein. For example, other cells,tissue, tissue fragments, growth factors such as VEGF and other knownangiogenic or arteriogenic growth factors, biologically active or inertcompounds, resorbable scaffolds, or other additives intended to enhancethe delivery, efficacy, tolerability, or function of the population ofregenerative cells may be added. The regenerative cell population mayalso be modified by insertion of DNA or by placement in a cell culturesystem (as described herein or known in the art) in such a way as tochange, enhance, or supplement the function of the regenerative cellsfor derivation of a structural or therapeutic purpose. For example, genetransfer techniques for stem cells are known by persons of ordinaryskill in the art, as disclosed in (Morizono et al., 2003; Mosca et al.,2000), and may include viral transfection techniques, and morespecifically, adeno-associated virus gene transfer techniques, asdisclosed in (Walther and Stein, 2000) and (Athanasopoulos et al.,2000). Non-viral based techniques may also be performed as disclosed in(Muramatsu et al., 1998). A gene encoding one or more cellulardifferentiating factors, e.g., a growth factor(s) or a cytokine(s),could also be added. Examples of various cell differentiation agents aredisclosed in (Gimble et al., 1995; Lennon et al., 1995; Majumdar et al.,1998; Caplan and Goldberg, 1999; Ohgushi and Caplan, 1999; Pittenger etal., 1999; Caplan and Bruder, 2001; Fukuda, 2001; Worster et al., 2001;Zuk et al., 2001). Genes encoding anti-apoptotic factors or agents couldalso be added. Addition of the gene (or combination of genes) could beby any technology known in the art including but not limited toadenoviral transduction, “gene guns,” liposome-mediated transduction,and retrovirus or lentivirus-mediated transduction, plasmid,adeno-associated virus. These regenerative cells could then be implantedalong with a carrier material bearing gene delivery vehicle capable ofreleasing and/or presenting genes to the cells over time such thattransduction can continue or be initiated in situ.

When the cells and/or tissue containing the cells are administered to apatient other than the patient from whom the cells and/or tissue wereobtained, one or more immunosuppressive agents may be administered tothe patient receiving the cells and/or tissue to reduce, and preferablyprevent, rejection of the transplant. As used herein, the term“immunosuppressive drug or agent” is intended to include pharmaceuticalagents which inhibit or interfere with normal immune function. Examplesof immunosuppressive agents suitable with the methods disclosed hereininclude agents that inhibit T-cell/B-cell costimulation pathways, suchas agents that interfere with the coupling of T-cells and B-cells viathe CTLA4 and B7 pathways, as disclosed in U.S. Patent Pub. No.20020182211. A preferred immunosuppressive agent is cyclosporine A.Other examples include myophenylate mofetil, rapamicin, andanti-thymocyte globulin. In one embodiment, the immunosuppressive drugis administered with at least one other therapeutic agent. Theimmunosuppressive drug is administered in a formulation which iscompatible with the route of administration and is administered to asubject at a dosage sufficient to achieve the desired therapeuticeffect. In another embodiment, the immunosuppressive drug isadministered transiently for a sufficient time to induce tolerance tothe regenerative cells of the invention.

In these embodiments, the regenerative cells may be contacted, combined,mixed or added to the additives through any art recognized manner,including devices such as the agitation devices and associated methodsdescribed herein. For example, rocking, inversion, compression pulsed ormoving rollers may be used.

In another aspect, the cell population could be placed into therecipient and surrounded by a resorbable plastic sheath or othermaterials and related components such as those manufactured by MacroPoreBiosurgery, Inc. (see e.g., U.S. Pat. Nos. 6,269,716; 5,919,234;6,673,362; 6,635,064; 6,653,146; 6,391,059; 6,343,531; 6,280,473).

In all of the foregoing embodiments, at least a portion of the separatedand concentrated regenerative cells may be cryopreserved, as describedin U.S. patent application Ser. No. 10/242,094, entitled PRESERVATION OFNON EMBRYONIC CELLS FROM NON HEMATOPOIETIC TISSUES, filed Sep. 12, 2002,which claims the benefit of U.S. Provisional Patent Application60/322,070 filed Sep. 14, 2001, which is commonly assigned, and thecontents of which in their entireties are expressly incorporated hereinby reference.

At the end of processing, the regenerative cells may be manuallyretrieved from the output chamber. The cells may be loaded into adelivery device, such as a syringe, for placement into the recipient byeither, subcutaneous, intramuscular, or other technique allowingdelivery of the cells to the target site within the patient. In otherwords, cells may be placed into the patient by any means known topersons of ordinary skill in the art. Preferred embodiments includeplacement by needle or catheter, or by direct surgical implantation. Inother embodiments, the cells may be automatically transported to anoutput chamber which may be in the form of a container, syringe orcatheter etc., which may be used to place the cells in the patient. Thecontainer may also be used to store the cells for later use or forcryopreservation. All retrieval methods are performed in a sterilemanner. In the embodiment of surgical implantation, the cells could beapplied in association with additives such as a preformed matrix orscaffold as described herein.

In preferred embodiments of the invention (e.g., the embodiment shown inFIG. 4), the system is automated. In another embodiment, the system hasboth automated and manual components. The system may be comprised of oneor more disposable components connected to or mounted on a re-usablehardware component or module. The automated systems of the inventionprovide screen displays (see FIG. 16) that prompt proper operation ofthe system. The automated systems may also provide a screen thatprovides status of the procedure and/or the step by step instructions asto the proper setup of the disposable components of the system. Thescreen may also indicate problems or failures in the system if theyoccur and provide “troubleshooting” guidance if appropriate. In oneembodiment, the screen is a user interface screen that allows the userto input parameters into the system through, e.g., a touch screen.

The partial and fully automated systems may include a processing device(e.g., microprocessor or personal computer) and associated softwareprograms that provide the control logic for the system to operate and toautomate one or more steps of the process based on user input. Incertain embodiments, one or more aspects of the system may beuser-programmable via software residing in the processing device. Theprocessing device may have one or more pre-programmed software programsin Read Only Memory (ROM). For example, the processing device may havepre-programmed software tailored for processing blood, another programfor processing adipose tissue to obtain small volumes of regenerativecells and another program for processing adipose tissue to obtain largervolumes of regenerative cells. The processing device may also havepre-programmed software which provides the user with appropriateparameters to optimize the process based on the user's input of relevantinformation such as the amount of regenerative cells required, the typeof tissue being processed, the type of post-processing manipulationrequired, the type of therapeutic application, etc.

The software may also allow automation of steps such as controlling theingress and egress of fluids and tissues along particular tubing pathsby controlling pumps and valves of the system; controlling the propersequence and/or direction of activation; detecting blockages withpressure sensors; mixing mechanisms, measuring the amount of tissueand/or fluid to be moved along a particular pathway using volumetricmechanisms; maintaining temperatures of the various components usingheat control devices; and integrating the separation and concentrationprocess with timing and software mechanisms. The processing device canalso control centrifuge speeds based on the tissue type being processedand/or the cell population or sub-population being harvested, and thetypes of procedures to be performed (e.g., tissue enhancement usingadipose tissue augmented with regenerative cells, or processing of cellsfor bone repair applications using regenerative cell coated bonegrafts). The processing device may also include standard parallel orserial ports or other means of communicating with other computers ornetworks. Accordingly, the processing device can be a stand alone unitor be associated one or more additional devices for the furtherprocessing methods described herein.

The software may allow for automated collection of “run data” including,for example, the lot numbers of disposable components, temperature andvolume measurements, tissue volume and cell number parameters, dose ofenzyme applied, incubation time, operator identity, date and time,patient identity, etc. In a preferred embodiment of the device acharacter recognition system, such as a bar code reading system would beintegrated to permit data entry of these variables (for exampledisposable set lot number and expiration date, lot number and expirationdate of the Collagenase, patient/sample identifiers, etc.) into theprocessing device as part of documentation of processing. This wouldreduce the opportunity for data entry errors. Such a bar code readingsystem could be easily incorporated into the processing device using aUSB or other interface port and system known to the art. In this way thedevice would provide integrated control of the data entry anddocumentation of the process. A print-out report of these parameterswould be part of the user-defined parameters of a programmed operationof the system. Naturally this would require integration of a printercomponent (hardware and driver) or printer driver in software plus aninterface output connector for a printer (e.g., a USB port) in thehardware of the device.

In certain embodiments, the system is a fully automated system. Forexample, the user may initially select the amount of tissue to beprocessed, attach the system to the patient and the system mayautomatically aspirate the required tissue and separate and concentrateregenerative cells in an uninterrupted sequence without further userinput. The user may also input the amount of regenerative cells requiredand allow the system to aspirate the requisite amount of tissue andprocess the tissue. A fully automated system also includes a systemwhich is capable of being reconfigured based on a number of (e.g., twoor more) user input parameters, e.g., number of wash cycles, speed ofcentrifugation etc. The system can also be run in semi-automatic modeduring which the system goes through certain steps without userintervention but requires user intervention before certain processes canoccur. In other embodiments, the system is a single integrated systemthat displays instructions to guide the user to perform predeterminedoperations at predetermined times. For example, the processing devicemay prompt users through the steps necessary for proper insertion oftubing, chambers and other components of the system. Accordingly, theuser can ensure that the proper sequence of operations is beingperformed. Such a system can additionally require confirmation of eachoperational step by the user to prevent inadvertent activation ortermination of steps in the process. In a further embodiment, the systemmay initiate automated testing to confirm correct insertion of tubing,chambers, absence of blockages etc. In yet another embodiment, thesystem of the present invention is capable of being programmed toperform multiple separation and concentration processes throughautomated control of tissue flow through the system. This feature may beimportant, for example, during surgery on a patient where tissue thatwould otherwise be lost is collected into the system, and regenerativecells from the tissue are separated and concentrated and returned to thepatient.

As set forth above, components of the system may be disposable (referredto herein as “disposable set(s)”), such that portions of the system canbe disposed of after a single use. This implementation can help ensurethat any surface which comes in contact with the patient's tissue willbe disposed of properly after being used. An exemplary disposable set isillustrated in FIG. 13. In a preferred embodiment, the disposablecomponents of the system are pre-sterilized and packaged so as to beusable “off the shelf” that are easy to use and easy to load and thateliminate the need for many tubing connections and complex routing oftubing connections. Such disposable components are relativelyinexpensive to manufacture, and therefore, do not create a substantialexpense due to their disposal. In one embodiment, the disposable system(referred to interchangeably herein as “disposable set(s)”) comprises,consists essentially of, or consists of, the collection chamber 20, theprocessing chamber 30, the waste chamber 40, the output chamber 50, thefilter assemblies 36, the sample bag 60 and the associated conduits 12or tubing. In preferred embodiments of the disposable sets of thesystem, the collection chamber 20 and the processing chamber 30 areconnected by way of conduits 12 that are housed in a rigid frame. Therotating seal network (FIGS. 7 & 8) of a processing chamber 30 may alsobe housed in the same rigid frame. In another preferred embodiment, thevarious chambers and containers of the disposable set are comprised ofthe necessary interfaces that are capable of communicating with theprocessing device of the system such that the pumps, valves, sensors andother devices that automate the system are appropriately activated orde-activated as needed without user intervention. The interfaces alsoreduce the time and expertise required to set up the system and alsoreduce errors by indicating how to properly set up the system andalerting the user in the event of an erroneous setup.

Most of the disposable sets of the invention will have many commonelements. However, the ordinarily skilled artisan will recognize thatdifferent applications of the system may require additional componentswhich may be part of the disposable sets. Accordingly, the disposablesets may further comprise one or more needles or syringes suitable forobtaining adipose or other tissue from the patient and returningregenerative cells to the patient. The type number and variety of theneedles and syringes included will depend on the type and amount oftissue being processed. The disposable sets may further comprise one ormore rigid or flexible containers to hold washing fluids and otherprocessing reagents used in the system. For example, the disposable setsmay comprise containers to hold saline, enzymes and any other treatmentor replacement fluids required for the procedure. In addition, suitablewashing solutions, re-suspension fluids, additives, agents or transplantmaterials may be provided with the disposable sets for use inconjunction with the systems and methods of the invention.

Any combination of system components, equipment or supplies describedherein or otherwise required to practice the invention may be providedin the form of a kit. For example, a kit of the invention may include,e.g., the optimal length and gage needle for the syringe basedliposuction and sterile syringes which contain the preferred filtermedia which allows for the processing of small volumes of tissue. Otherexemplary equipment and supplies which may be used with the inventionand may also be included with the kits of the invention are listed inTables II and III.

Table II below identifies examples of supplies that can be used in toobtain adipose derived regenerative cell in accordance with the systemsand methods of the present invention:

TABLE II Description Vendor Quantity Note 10 ml syringe Becton-Dickinsonas req'd Optional, used for liposuction 14GA blunt tip needle as req'dOptional, used for liposuction Single Blood Pack Baxter Fenwal 1 Maincell processing bag; bag has (600 ml) spike adaptor on line and two freespike ports Transfer pack with Baxter Fenwal 1 Quad bag set coupler (150ml) Transfer pack with Baxter Fenwal 1 Waste bag coupler (1 L) SampleSite Coupler Baxter Fenwal 2 0.9% saline (for Baxter Fenwal 1 injection)14GA sharp needle Monoject as req'd For adding liposuction tissue to bag20GA sharp needle Monoject 3 For adding collagenase and removing PLAcells 0.2 μm Sterflip filter Millipore 1 For filtering collagenaseTeruflex Aluminium Terumo 4 ME*ACS121 for temporary sealing clips tubesealing Povidone Iodine prep Triadine as req'd 10-3201 pad Liberase H1Roche See Procedure Note 1 Collagenase TSCD wafers Terumo 2 1SC*W017 foruse with TSCD Sterile Tubing Welder

Table III, below, identifies equipment that may be used with the systemsand methods disclosed herein.

TABLE III Description Vendor Quantity Note Sorvall Legend T Easy Fisher1 75-004-367 Set Centrifuge Scientific Rotor Kendro/Sorvall 1 TTH-750rotor Rotor buckets Kenro/Sorvall 4 75006441 round buckets Adaptor for150 ml bags Kendro/Sorvall 4 00511 Plasma Expressor Baxter Fenwal 14R4414 Tube Sealer Sebra 1 Model 1060 TSCD Sterile Tubing Terumo 13ME*SC201AD Welder LabLine Thermal Rocker LabLine 1 4637 ‘Disposable’plastic Davron 3 hemostat-style clamp Balance Bags Sets 2 Water-filledbags used to balance centrifuge Biohazard Sharps 1 Chamber BiohazardWaste 1 Chamber

The re-usable component of the system comprises, consists essentiallyof, or consists of the agitation mechanism for the collection chamber,the pump, and assorted sensors which activate valves and pump controls,the centrifuge motor, the rotating frame of the centrifuge motor, theuser interface screen and USB ports, an interlocking or docking deviceor configuration to connect the disposable set such that the disposableset is securely attached to and interface with the re-usable hardwarecomponent and other associated devices. An exemplary re-usable componentis illustrated in FIG. 14. In preferred embodiments, the re-usablecomponent includes a means for separating and concentrating theregenerative cells from the regenerative cell composition, e.g., arotating centrifuge. In this embodiment, the re-usable component isdesigned connect to and interface with a portion of the processingchamber (comprising a centrifuge chamber) of the disposable set as shownin FIG. 15A. It is understood that the means for separating andconcentrating regenerative cells in the re-usable component is notlimited to a rotating centrifuge but may also include any otherconfiguration described herein, including a spinning membrane filter.The re-usable component may also house the processing device describedherein which contains pre-programmed software for carrying out severaldifferent tissue processing procedures and selectively activating thevarious pumps and valves of the system accordingly. The processor mayalso include data storage capability for storing donor/patientinformation, processing or collection information and other data forlater downloading or compilation. The re-usable component may be usedwith a variety of disposable sets. The disposable set is connected tothe re-usable component through, e.g., an interlocking device orconfiguration to connect the disposable set such that the disposable setis securely attached to and interfaces with the re-usable hardwarecomponent in a manner that the processing device present on there-usable component can control, i.e., send and receive signals to andfrom the various components of the disposable set as well as variouscomponents of the re-usable component and other associated devices andsystems.

In one embodiment, a disposable set for use in the system is comprisedof a collection chamber 20 which can accommodate about 800 mL of tissue;a processing chamber 30 which can process the regenerative cellcomposition generated by about 800 mL of tissue washed and digested inthe collection chamber 20; an output chamber 50 which can accommodate atleast 0.5 mL of regenerative cells; and a waster container 40 which canaccommodate about 10 L of waste. In this embodiment, the hardware deviceis no larger than 24″L×18″W×36″H. Alternative dimensions of the variouscomponents of the disposable sets as well as the hardware device may beconstructed as needed and are intended to be encompassed by the presentinvention without limitation.

The disposable components of the system are easy to place on the device.An illustration of a disposable set utilized assembled together with acorresponding re-usable component is illustrated in FIG. 15A. The systemis preferably designed such that it can detect an improperly loadeddisposable component. For example, the components of each disposable setmay have color-guided marks to properly align and insert the tubing,chambers etc. into appropriate places in the system. In additionalembodiments, the system disclosed herein is a portable unit. Forexample, the portable unit may be able to be moved from one locationwhere adipose tissue harvesting has occurred, to another location foradipose tissue harvesting. In certain implementations, the portable unitis suitable for harvesting and processing of adipose tissue by apatient's bedside. Thus, a portable unit may be part of a system whichcan be moved from patient to patient. Accordingly, the portable unit maybe on wheels which lock in place and, thus, can be easily placed andused in a convenient location in a stable and secure position throughoutthe procedure. In other embodiments, the portable unit is designed forset-up and operation on a flat surface such as a table top. The portableunit may also be enclosed in a housing unit. The portable unit mayfurther be comprised of hangers, hooks, labels, scales and other devicesto assist in the procedure. All of the herein described re-usablecomponents of the system such as the centrifuge, processing device,display screen may be mounted on the portable unit of the system.

Alternate manual embodiments for obtaining regenerative cells are alsowithin the scope of this invention. For example, in one embodiment,adipose tissue may be processed using any combination of the componentsof the system, equipment and/or supplies described herein.

A particular example of the system embodying the present invention isshown in FIG. 4. FIG. 4 illustrates an automated system and method forseparating and concentrating regenerative cells from tissue, e.g.,adipose tissue, suitable for re-infusion within a patient. In certainembodiments of the system shown in FIG. 4, the system further includesan automated step for aspirating a given amount of tissue from thepatient. The system shown in FIG. 4 is comprised of the disposable setshown in FIG. 13 which is connected to the re-usable component of thesystem shown in FIG. 14 to arrive at an automated embodiment of thesystem shown in FIG. 15A. The disposable set is connected to there-usable component through, e.g., an interlocking or docking device orconfiguration, which connects the disposable set to the re-usablecomponent such that the disposable set is securely attached to andassociated with the re-usable hardware component in a manner that theprocessing device present on the re-usable component can control andinterface with, i.e., send and receive signals to and from the variouscomponents of the disposable set as well as various components of there-usable component and other associated devices and systems.

The user may connect the disposable set to the re-usable component,input certain parameters using the user interface, e.g., the volume oftissue being collected, attach the system to the patient, and the systemautomatically performs all of the steps shown in FIG. 4 in anuninterrupted sequence using pre-programmed and/or user inputparameters. One such sequence is illustrated in FIG. 15B. Alternatively,the tissue may be manually aspirated from the patient by the user andtransported to system for processing, i.e., separation and concentrationof regenerative cells.

Specifically, as shown in FIG. 4, tissue, e.g., adipose tissue, may bewithdrawn from the patient using conduit 12 and introduced intocollection chamber 20. A detailed illustration of the collection chamberof FIG. 4 is shown in FIG. 5. As illustrated in FIG. 5, the collectionchamber 20 may be comprised of a vacuum line 11 which facilitates tissueremoval using a standard cannula. The user may enter the estimatedvolume of tissue directed to the collection chamber 20 at this point.The tissue is introduced into the collection chamber 20 through an inletport 21 which is part of a closed fluid pathway that allows the tissue,saline and other agents to be added to the tissue in an aseptic manner.An optical sensor of the system, e.g., sensor 29, can detect when theuser input volume of tissue is present in the collection chamber 20. Incertain embodiments, if less tissue is present in the collection chamberthan the user input, the user will have the option to begin processingthe volume of tissue which is present in the collection chamber 20. Incertain embodiments, a portion of the tissue removed from the patientmay be directed to the sample chamber 60 through the use of a pump,e.g., a peristaltic pump, via a conduit, which may be activated via userinput utilizing the user interface.

A sensor 29 can signal the processing device present in the re-usablecomponent to activate the steps needed to wash and disaggregate thetissue. For example, the processing device may introduce a pre-setvolume of washing agent based on the volume of tissue collected usingautomated valves and pumps. This cycle may be repeated in the collectionchamber until the optical sensor determines that the effluent liquid issufficiently clear and devoid of unwanted material. For example, anoptical sensor 29 along the conduit leading out of the collectionchamber 12 b or 12 d can detect that the unwanted materials have beenremoved and can signal the processing device to close the requiredvalves and initiate the next step.

Next, the processing device may introduce a pre-programmed amount ofdisaggregation agent based on the volume of tissue collected. Theprocessing device may also activate agitation of the tissue in thecollection chamber for a preset period of time based on the initialvolume of tissue collected or based on user input. In the embodimentshown in FIG. 4, once the disaggregation agent, e.g., collagenase, isadded to the collection chamber 20 through the collagenase source 24,the motor in the collection chamber 20 is activated via the processingdevice. The motor activates the rotatable shaft 25 which is comprised ofa magnetic stirrer and a paddle-like device wherein one or more paddles25 a are rigidly attached to the filter cage 27 of a filter prefixed tothe collection chamber 28. The paddles agitate the in the presence ofthe disaggregation agent such that the regenerative cells separate fromthe tissue.

The solution in the collection chamber 20 is allowed to settle for apreset period of time. The buoyant portion of the solution is allowed torise to the top of the solution. Once the preset period of time elapses,the necessary valves and pumps are activated by the processing device toremove the non-buoyant portion to the waste chamber 40. The transferinto the waste chamber 40 continues until a sensor 29 along the conduitleading out of the collection chamber 12 b or 12 d can detect that thebuoyant fraction of the solution is about to be transferred to the wastechamber 30. For example, a sensor 29 along the conduit leading out ofthe collection chamber 12 b or 12 d can detect that the unwantedmaterials have been removed and can signal the processing device toclose the required valves.

At this time the non-buoyant fraction of the solution, i.e., theregenerative cell composition, is moved to the processing chamber 30.This is accomplished through the use of the necessary valves andperistaltic pumps. In certain embodiments, before transfer of theregenerative cell composition to the processing chamber 30, anadditional volume of saline may be added to the buoyant fraction ofsolution remaining in the collection chamber 20. Another wash cycle maybe repeated. After this cycle, the solution is allowed to settle and thenon-buoyant fraction (which contains the regenerative cells) istransported to the processing chamber 30 and the buoyant fraction isdrained to the waste chamber 40. The additional wash cycle is used tooptimize transfer of all the separated regenerative cells to theprocessing chamber 30.

Once the regenerative cell composition is transported to the processingchamber 30 by way of conduits 12, the composition may be subject to oneor more additional washing steps prior to the start of the concentrationphase. This ensures removal of waste and residual contaminants from thecollection chamber 20. Similarly, subsequent to the concentration step,the regenerative cell composition may be subjected to one or moreadditional washing steps to remove residual contaminants. The unwantedmaterials may be removed from the processing chamber 30 to the wastechamber 40 in the same manner, i.e., control of valves and pumps viasignals from the processing device, as described above.

The various embodiments of the processing chamber 30 shown in FIG. 4 aredescribed in detail below. The processing chamber 30 shown in FIG. 4 isin the form of a centrifuge chamber. A detailed illustration of theprocessing chamber of FIG. 4 is shown in FIGS. 7 and 8. Such aprocessing chamber 30 is generally comprised of a rotating seal network30.1 comprising an outer housing 30.2, one or more seals 30.3, one ormore bearings 30.4 and an attachment point 30.6 for connecting theprocessing chamber to the centrifuge device present in the re-usablecomponent of the system; one or more fluid paths 30.5 in the form ofconduits extending out from the rotating seal and ending in a centrifugechamber on each end which is in the form of an output chamber 50 housedin a frame 53 wherein the frame is comprised of one or more ports 52 andone or more handles to manually re-position the output chamber 50.

The rotating seal network 30.1 is included to ensure that the fluidpathways of the processing chamber can be maintained in a sterilecondition. In addition, the fluid pathways of the processing chamber canbe accessed in a sterile manner (e.g., to add agents or washingsolution) at any time, even while the centrifuge chamber of theprocessing chamber is spinning.

The rotating seal network 30.1 shown in FIGS. 7 and 8 includes arotating shaft comprised of two or more bearings 30.4, three or more lipseals 30.3, and an outer housing 30.2. In this embodiment, the bearings30.4 further comprise an outer and inner shaft (not shown) referred toherein as races. These races may be separated by precision groundspheres. The races and spheres comprising the bearings are preferablyfabricated with material suitable for contact with bodily fluid, or arecoated with material suitable for contact with bodily fluid. In apreferred embodiment, the races and spheres are fabricated using, forexample, silicone nitride or zirconia. Furthermore, in this embodiment,the three lip seals are comprised of a circular “U” shaped channel (notshown) as well as a circular spring (not shown). The circular “U” shapedchannel is preferably fabricated using flexible material such that aleakage proof junction with the rotating shaft of the rotating sealnetwork 30.1 is formed. Additionally, the lip seals are preferablyoriented in a manner such that pressure from the regenerative cellcomposition flowing through the processing chamber causes the sealassembly to tighten its junction with the rotating shaft by way ofincreased tension. The seals may be secured in position by way of one ormore circular clips (not shown) which are capable of expanding and/orcollapsing as needed in order to engage a groove in the outer housing30.2 of the rotating seal network 30.1. The heat generated by or nearthe rotating seal network 30.1 must be controlled to prevent lysis ofthe cells in the solution which is being moved through the passage. Thismay be accomplished by, for example, selecting a hard material forconstructing the rotating shaft, polishing the area of the rotatingshaft which comes in contact with the seals and minimizing contactbetween the rotating shaft and the seal.

In another embodiment the rotating seal network 30.1 is comprised of asingle rubber seal 30.3 and an air gasket (not shown). This seal andgasket provide a tortuous path for any biologic matter which couldcompromise the sterility of the system. In another embodiment therotating seal network 30.1 is comprised of multiple spring loaded seals30.3 which isolate the individual fluid paths. The seals 30.3 arefabricated of a material which can be sterilized as well as seal therotating shaft without lubricant. In another embodiment the rotatingseal network 30.1 is compromised of a pair of ceramic disks (not shown)which create the different fluid paths and can withstand the rotation ofthe system and not cause cell lysis. In another embodiment the fluidpathway is flexible and is allowed to wind and unwind with respect tothe processing chamber. This is accomplished by having the flexiblefluid pathway rotate one revolution for every two revolutions of theprocessing chamber 30. This eliminates the need for a rotating sealaltogether.

The regenerative cell composition is pumped from the collection chamber20 along a fluid path through the axis of rotation of the rotating sealnetwork 30.1 and then divides into a minimum of two fluid pathways 30.5each of which radiate outward from the central axis of the processingchamber 30 and terminate near the outer ends of the processing chamber30, i.e., within the centrifuge chambers which house the output chambers50 (FIGS. 7 and 8). Accordingly, in a preferred embodiment, theprocessing chamber 30 is comprised of two or more output chambers 50 asshown in FIGS. 7 and 8. These output chambers 50 are positioned suchthat they are in one orientation during processing 30.7 and anotherorientation for retrieval of concentrated regenerative cells 30.8. Forexample, the output changes are tilted in one angle during processingand another angle for cell retrieval. The cell retrieval angle is morevertical than the processing angle. The two positions of the outputchamber 50 may be manually manipulated through a handle 53 whichprotrudes out of the processing chamber 30. The regenerative cells canbe manually retrieved from the output chambers 50 when they are in theretrieval orientation 30.8 using a syringe. In another embodiment, fluidpath 30.5 is constructed such that it splits outside the processingchamber and then connects to the outer ends of the processing chamber30, i.e., within the centrifuge chambers which house the output chambers50 (not shown). In this embodiment, large volumes of regenerative cellcomposition and/or additives, solutions etc. may be transported to thecentrifuge chamber and/or the output chambers directly.

With reference to FIGS. 4 and 7-9, between the collection chamber 20 andthe processing chamber 30, a pump 34 and one or more valves 14 may beprovided. In a preferred embodiment, the valves 14 are electromechanicalvalves. In addition, sensors, such as pressure sensor 29, may beprovided in line with the processing chamber 30 and the collectionchamber 20. The valves, pumps and sensors act in concert with theprocessing device present on the re-usable component (FIG. 14) toautomate the concentration steps of the system.

The sensors detect the presence of the regenerative cell composition inthe centrifuge chambers and activate the centrifuge device throughcommunication with the processing device of the system. The regenerativecell composition is then subjected to a pre-programmed load for apre-programmed time based on the amount of tissue originally collectedand/or user input. In certain embodiments, this step may be repeatedeither automatically or through user input. For example, the compositionis subjected to a load of approximately 400 times the force of gravityfor a period of approximately 5 minutes. The output chamber 50 isconstructed such that the outer extremes of the chamber form a smallreservoir for the dense particles and cells. The output chamber 50retains the dense particles in what is termed a ‘cell pellet’, whileallowing the lighter supernatant to be removed through a fluid path,e.g., a fluid path which is along the axis of rotation of the rotatingseal network 30.1 and travels from the low point in the center of theprocessing chamber 30 through the rotating seal network 30.1 to thewaste container 40. The valves 14 and pumps 34 signal the processingdevice to activate steps to remove the supernatant to the wastecontainer 40 without disturbing the cell pellet present in the outputchamber 50.

The cell pellet that is obtained using the system shown in FIG. 4comprises the concentrated regenerative cells of the invention. In someembodiments, after the supernatant is removed and directed to the wastechamber 40, a fluid path 30.5 may be used to re-suspend the cell pelletthat is formed after centrifugation with additional solutions and/orother additives. Re-suspension of the cell pellet in this manner allowsfor further washing of the regenerative cells to remove unwantedproteins and chemical compounds as well as increasing the flow of oxygento the cells. The resulting suspension may be subjected to another loadof approximately 400 times the force of gravity for another period ofapproximately 5 minutes. After a second cell pellet is formed, and theresulting supernatant is removed to the waste chamber 40, a final washin the manner described above may be performed with saline or some otherappropriate buffer solution. This repeated washing can be performedmultiple times to enhance the purity of the regenerative cell solution.In certain embodiments, the saline can be added at any step as deemednecessary to enhance processing. The concentrations of regenerativecells obtained using the system shown in FIG. 4 may vary depending onamount of tissue collected, patient age, patient profile etc. Exemplaryyields are provided in Table 1.

The final pellet present in the output chamber 50 may then be retrievedin an aseptic manner using an appropriate syringe after the outputchamber 50 is positioned in the orientation appropriate for cellremoval. In other embodiments, the final pellet may be automaticallymoved to a container in the in the output chamber 50 which may beremoved and stored or used as needed. This container may be in anyappropriate form or size. For example, the container may be a syringe.In certain embodiments, the output container 50 itself may be heatsealed (either automatically or manually) and isolated from the othercomponents of the processing chamber for subsequent retrieval and use ofthe regenerative cells in therapeutic applications as described hereinincluding re-infusion into the patient. The cells may also be subject tofurther processing as described herein either prior to retrieval fromthe output chamber or after transfer to a second system or device. There-usable component shown in FIG. 14 is constructed such that it can beconnected to one or more additional systems or devices for furtherprocessing as needed.

2. Methods of Manufacturing a Cell Carrier Portion of the Device

The cell carrier portion of the present invention is critical formaintaining the bone forming functions of the regenerative cells.Specifically, the cell carrier, on which the regenerative cells areplaced, as further described herein, serves to organize the cells inthree dimensions. Accordingly, in considering substrate materials, it isimperative to choose one that exhibits clinically acceptablebiocompatibility. In addition, the mechanical properties of the cellcarrier must be sufficient so that it does not collapse during thepatient's normal activities.

A variety of cell carriers are known and used in the art and areintended to be encompassed by the present invention. Cell carriers inthe form of gels, such as hydrogels are encompassed by the presentinvention. Fiber based cell carriers are also encompassed by the presentinvention and include woven meshes, knitted meshes, non-woven meshes(felts) and polymer coated meshes (to enhance strength). Methods formanufacture fiber based cell carriers are known in the art. For example,cell carriers based on knitted meshes can be made according to themethods described by H. J. Buchsbaum, W. Christopherson, S. Lifshitz,and S. Bernstein. Vicryl mesh in pelvic floor reconstruction. Arch.Surg120 (12):1389-1391, 1985. Non-woven mesh based cell carriers can be madeaccording to the methods described by L. E. Freed, G. Vunjak-Novakovic,R. J. Biron, D. B. Eagles, D. C. Lesnoy, S. K. Barlow, and R. Langer.Biodegradable polymer cell carriers for tissue engineering.Biotechnology (N Y) 12 (7):689-693, 1994. Polymer coated meshes (toenhance strength) can be made according to the methods described by W.S. Kim, J. P. Vacanti, L. Cima, D. Mooney, J. Upton, W. C. Puelacher,and C. A. Vacanti. Cartilage engineered in predetermined shapesemploying cell transplantation on synthetic biodegradable polymers.Plast Reconstr Surg 94 (2):233-237, 1994.

In a preferred embodiment, the cell carrier portion of the device willbe preformed into particulates. The sizes and shapes of theseparticulates can range from less than 1 mm to up to 10 mm in diameterand from abstract shaped granules to defined shapes, respectively. Theseparticulates can either be highly porous (greater than 95% void volume)or non-porous. The cell carrier particulates can be used to contain thecells into the center of or around the outside of the containment deviceand can be preloaded into or onto the containment device beforepackaging and sterilization or can be packaged and sterilized separatelyfrom the containment device.

Sponge or foam based cell carriers are also encompassed by the presentinvention and can be manufactured using methods such as solventcasted-particulate leached (SC-PL), melt molded-particulate leached(where melt molding can include e.g., compression molding, injectionmolding or extrusion), extrusion-particulate leaching, emulsion-freezedrying, freeze drying combined with particulate-leaching, solutioncasting, gel casting, atomized foams, phase separation, phase separationcombined with particulate leaching, high pressure CO2, gas foaming ofeffervescent salts, 3D printing, fused deposition modeling and methodsusing electrospun nanofibers or other nanofibers. In a preferredembodiment, the cell carrier portion of the present invention ismanufactured using solvent casting/freeze drying methods.

Methods for manufacturing the sponge or foam based cell carrier of theinvention are known in the art. For example, solvent casted-particulateleached (SC-PL) cell carriers can be made according to the methodsdisclosed in A. G. Mikos, A. J. Thorsen, L. A. Czerwonka, Bao Y., and R.Langer. Preparation and characterization of poly(L-lactic acid) foams.Polymer 35 (5):1068-1077, 1994 and P. X. Ma and J. W. Choi.Biodegradable polymer cell carriers with well-defined interconnectedspherical pore network. Tissue Eng 7 (1):23-33, 2001. SC-melt moldedcell carriers can be made according to R. C. Thomson, M. J. Yaszemski,J. M. Powers, and A. G. Mikos. Hydroxyapatite fiber reinforcedpoly(alpha-hydroxy ester) foams for bone regeneration. Biomaterials 19(21):1935-43, 1998. Extrusion-particulate leaching based cell carrierscan be made according to M. S. Widmer, P. K. Gupta, L. Lu, R. K.Meszlenyi, G. R. Evans, K. Brandt, T. Savel, A. Gurlek, C. W. Patrick,Jr., and A. G. Mikos. Manufacture of porous biodegradable polymerconduits by an extrusion process for guided tissue regeneration.Biomaterials 19 (21): 1945-1955, 1998.

Freeze-drying (Phase separation) based cell carriers can be madeaccording to H. Lo, Ponticiello M. S., and K. W. Leong. Fabrication ofcontrolled release biodegradable foams by phase separation. Tissue Eng1(1):15-28, 1995 and C. Schugens, V. Maquet, C. Grandfils, R. Jerome,and P. Teyssie. Polylactide macroporous biodegradable implants for celltransplantation. II. Preparation of polylactide foams by liquid-liquidphase separation. J Biomed Mater. Res 30 (4):449-461, 1996. Scaffoldsmanufactured using freeze drying (Phase separation) combined withparticulate-leaching can be made according to J. H. de Groot, A. J.Nijenhuis, Bruin P., A. J. Pennings, R. P. H. Veth, J. Klompmaker, andH. W. B. Jansen. Use of porous biodegradable polymer implants inmeniscus reconstruction. 1. Preparation of porous biodegradablepolyurethanes for the reconstruction of meniscus lesions. Colloid Polym.Sci. 268:1073-1081, 1990 and Q. Hou, D. W. Grijpma, and J. Feijen.Preparation of interconnected highly porous polymeric structures by areplication and freeze-drying process. J. Biomed Mater. Res 67B(2):732-740, 2003. Freeze-extraction based cell carriers can be madeaccording to M. H. Ho, P. Y. Kuo, H. J. Hsieh, T. Y. Hsien, L. T. Hou,J. Y. Lai, and D. M. Wang. Preparation of porous cell carriers by usingfreeze-extraction and freeze-gelation methods. Biomaterials 25(1):129-138, 2004. Emulsion—Freeze drying based cell carriers can bemade using the methods disclosed in K. Whang, C. H. Thomas, and K. E.Healy. A novel method to fabricate bioabsorbable cell carriers. Polymer36 (4):837-842, 1995.

Scaffolds manufactured using gel casting or solution casting methods canbe manufactured using J. P. Schmitz and J. O. Hollinger. A preliminarystudy of the osteogenic potential of a biodegradablealloplastic-osteoinductive alloimplant. Clin Orthop. (237):245-255,1988, A. G. Coombes and J. D. Heckman. Gel casting of resorbablepolymers. 1. Processing and applications. Biomaterials 13 (4):217-224,1992 and the methods disclosed in U.S. Pat. No. 5,716,416 (1998).Methods of manufacturing cell carriers using atomized foams can found inH. Lo, Ponticiello M. S., and K. W. Leong. Fabrication of controlledrelease biodegradable foams by phase separation. Tissue Eng 1(1):15-28,1995. Methods using gas Foaming-particulate leaching can be found in Y.S, Nam, J. J. Yoon, and T. G. Park. A novel fabrication method ofmacroporous biodegradable polymer cell carriers using gas foaming saltas a porogen additive. J Biomed Mater.Res 53 (1):1-7, 2000.

The 3D printing (Theriform™ process) is described in, for example, R. A.Giordano, B. M. Wu, S. W. Borland, L. G. Cima, E. M. Sachs, and M. J.Cima. Mechanical properties of dense polylactic acid structuresfabricated by three dimensional printing. J Biomater. Sci Polym. Ed 8(1):63-75, 1996 and S. S. Kim, H. Utsunomiya, J. A. Koski, B. M. Wu, M.J. Cima, J. Sohn, K. Mukai, L. G. Griffith, and J. P. Vacanti. Survivaland function of hepatocytes on a novel three-dimensional syntheticbiodegradable polymer cell carrier with an intrinsic network ofchannels. Ann Surg 228 (1):8-13, 1998. Fused deposition modeling basedmethods can be found in D. W. Hutmacher, T. Schantz, I. Zein, K. W. Ng,S. H. Teoh, and K. C. Tan. Mechanical properties and cell culturalresponse of polycaprolactone cell carriers designed and fabricated viafused deposition modeling. J Biomed Mater. Res 55 (2):203-216, 2001.Methods using electrospun nanofibers can be found in W. J. Li, C. T.Laurencin, E. J. Caterson, R. S. Tuan, and F. K. Ko. Electrospunnanofibrous structure: a novel cell carrier for tissue engineering. JBiomed Mater. Res 60 (4):613-621, 2002. Methods using nanofibers withporogen leaching can be found in R. Zhang and P. X. Ma. Syntheticnano-fibrillar extracellular matrices with predesigned macroporousarchitectures. J Biomed Mater. Res 52(2):430-438, 2000.

Both natural (e.g., collagen, elastin, poly(amino acids); andpolysaccharides such as hyaluronic acid, glycosamino glycan,carboxymethylcellulose; and ceramic based-cell carriers such as poroushydroxyapatite, tricalcium phosphate, and chitosan) and syntheticmaterials may be used to manufacture the cell carriers of the presentinvention. In a preferred embodiment, the cell carrier is constructed ofa resorbable material to allow room for tissue growth in the cellcarrier while eliminating the need for a second surgery to remove thecell carrier. Exemplary synthetic resorbable polymers that may be usedto manufacture the cell carriers of the present invention include,poly(glycolic acid) (PGA), poly(L-lactic acid) (PLLA), poly (D-lactide)(PDLA), poly (D,L-lactide)(PDLLA) and their copolymers, as well as PCL,PDO and PTMC and their co-polymers. These polymers offer distinctadvantages in that their sterilizability and relative biocompatibilityhave been well documented. Also, their resorption rates can be tailoredto match that of new tissue formation. In a preferred embodiment, thecell carrier is constructed of 70:30 poly(L-lactide-co-D,L-lactide). Inanother embodiment, the cell carrier is constructed of 85:15 poly(D,L-lactide-co-glycolide).

In certain embodiments, any of the scaffolds or cell carrier portionsdescribed above may be coated with apatite using a simulated body fluid(SBF) solution. The SBF solutions may be prepared with ionconcentrations approximately 0-10 times that of human blood plasma andcan be sterile filtered through a 0.22 μm PES membrane or a similarmembrane filter. Methods of making art-recognized SBF solutions andvariations thereof for use in the present invention can be found in,e.g., Chou et al. (2004) The Effect of Biomimetic Apatite Structure onOsteoblast Vitality, Proliferation and Gene Expression Biomaterials (Inpress); Oyane et al. (2003) Preparation and Assessment of RevisedSimulated Body Fluids J. Biomed mater Res 65A: 188-195; Murphy et al.(1999) Growth of Continuous Bonelike Mineral Within PorousPoly(lactic-co-glycolide) Scaffolds In Vitro J. Biomed. Mater. Res. 50:50-58. The scaffolds or cell carriers may be treated with glowdischarge, argon-plasma etching prior to being soaked in the SBFsolution to improve wettability and affinity for the SBF ions. Differentapatite microenvironments can be created on the scaffold or cell carriersurfaces by controlling the SBF concentration, components, pH and theduration of the scaffold or cell carrier in each SBF solution. Vacuum orfluid flow (directed or non-directed) can be used to force the SBF intothe pores of the scaffold or cell carrier portion.

It is understood in the art that desired resorption rates of the cellcarrier portion will vary based on the particular therapeuticapplication. It is believed that the cell carrier portion of the presentinvention having a thickness of 0.1 millimeters to about 5 millimetersshould maintain its structural integrity for a period in excess of abouttwo weeks to multiple months, preferably three to six months, beforesubstantially degrading, so that the bone forming can be achieved andoptimized.

The rates of resorption of the cell carrier may also be selectivelycontrolled. For example, the cell carrier portion may be manufactured todegrade at different rates depending on the rate of recovery of thepatient from a surgical procedure. Thus, a patient who recovers morequickly from a surgical procedure relative to an average patient, may beadministered an agent that for example is selective for the polymericmaterial of the cell carrier portion and causes the cell carrier portionto degrade more quickly. Or, if the polymeric material is, for example,temperature sensitive or is influenced by electrical charge, the area inwhich the device has been implanted may be locally heated or cooled, orotherwise exposed to an electrical charge that causes the device todissolve at a desired rate for the individual patient.

In addition to resorption rates, certain physical characteristics of thecell carriers must also be considered. Generally, the cell carrier musthave large a large surface area to allow cell attachment. One method ofachieving this result may be to create a highly porous polymer foam. Inthese foams, the pore size should be large enough so that cellspenetrate the pores, and the pores must be interconnected to facilitatenutrient exchange by cells deep within the construct. Thesecharacteristics (porosity and pore size) are often dependent on themethod of cell carrier fabrication (Mikos, A. G., Bao, Y., Cima, L. G.,Ingber, D. E., Vacanti, J. P. and Langer, R. (1993a). Preparation ofPoly(glycolic acid) bonded fiber structures for cell attachment andtransplantation. Journal of Biomedical Materials Research 27:183-189;Mikos, A. G., Sarakinos, G., Leite, S. M., Vacanti, J. P. and Langer, R.(1993b) Mikos, A. G., Thorsen, A. J., Czerwonka, L. A., Bao, Y., Langer,R., Winslow, D. N. and Vacanti, J. P. (1994). Preparation andcharacterization of poly(L-lactic acid) foams. Polymer 35:1068-1077;Nam, Y. S, and Park, T. G. (1999a). Biodegradable polymericmicrocellular foams by modified thermally induced phase separationmethod. Biomaterials 20:1783-1790; Nam, Y. S., Yoon, J. J. and Park, T.G. (2000). A novel fabrication method of macroporous biodegradablepolymer scaffolds using gas foaming salt as a porogen additive. Journalof Biomedical Materials Research (Applied Biomaterials) 53:1-7).

Optimal pore sizes for bone formation generally range from 40 to 400microns. Several methods have been developed to create highly porouscell carriers, including fiber bonding (Mikos et al. 1993 above),solvent casting/particulate leaching (Mikos et al. 1993 above; Mikos etal. 1994 above), gas foaming (Nam et al. 2000 above) and phaseseparation (Lo, H., Ponticiello, M. S, and Leong, K. W. (1995).Fabrication of controlled release biodegradable foams by phaseseparation. Tissue Engineering 1:15-28; Whang, K., Thomas, C. H., Healy,K. E. and Nuber, G. (1995). A novel method to fabricate bioabsorbablescaffolds. Polymer 36:837-842; Lo, H., Kadiyala, S., Guggino, S. E. andLeong, K. W. (1996). Poly(L-lactic acid) foams with cell seeding andcontrolled-release capacity. Journal of Biomedical Materials Research30:475-484; Schugens, C., Maquet, V., Grandfils, C., Jerome, R. andTeyssie, P. (1996). Polylactide macroporous biodegradable implants forcell transplantation II. Preparation of polylactide foams forliquid-liquid phase separation. Journal of Biomedical Materials Research30:449-461; Nam and Park 1999 above). Of these methods, fiber bonding,solvent casting/particulate leaching, gas foaming/particulate leachingand liquid-liquid phase separation produce large, interconnected poresto facilitate cell seeding and migration. The fiber bonding, solventcasting/particulate leaching and gas foaming/particulate leachingmethods exhibit clinically acceptable biocompatibility.

All of the methods described herein, as well as all art-recognizedmethods for manufacturing cell carriers, must be appropriately modifiedto remove organic solvents, which could reduce the ability of cells toform new tissues in vivo. Some methods of manufacturing the cellcarriers of the present invention are described herein by way of exampleand not limitation.

For example, as set forth above, one technique used for constructingthree-dimensional cell carriers is known as “melt molding,” wherein amixture of fine PLGA powder and gelatin microspheres is loaded in aTeflon® mold and heated above the glass-transition temperature of thepolymer. The PLGA-gelatin composite is removed from the mold and gelatinmicrospheres are leached out by selective dissolution in distilledde-ionized water. Other cell carrier manufacturing techniques includepolymer/ceramic fiber composite foam processing, phase separation, andhigh-pressure processing.

Another technique for manufacturing cell carriers is known as“solvent-casting and particulate-leaching.” In this technique, sievedsalt particles, such as sodium chloride crystals, are disbursed in aPLLA/chloroform solution which is then used to cast a membrane. Afterevaporating the solvent, the PLLA/salt composite membranes are heatedabove the PLLA melting temperature and then quenched or annealed bycooling at controlled rates to yield amorphous or semi-crystalline formswith regulated crystallinity. The salt particles are eventually leachedout by selective dissolution to produce a porous polymer matrix.

Yet another technique for manufacturing the cell carriers of the presentinvention is known as “fiber bonding”, and involves preparing a mold inthe shape of the desired cell carrier and placing fibers, such aspolyglycolic acid (PGA) into the mold and embedding the PGA fibers in apoly (L-lactic acid) (PLLA)/methylene chloride solution. The solvent isevaporated, and the PLLA-PGA composite is heated above the meltingtemperatures of both polymers. The PLLA is then removed by selectivedissolution after cooling, leaving the PGA fibers physically joined attheir cross-points without any surface or bulk of modifications andmaintaining their initial diameter. Fiber bonding is particularly usefulfor fabrication of thin cell carriers.

Another technique may be solid freeform fabrication (SFF) which refersto computer-aided-design and computer-aided-manufacturing (CAD/CAM)methodologies which have been used in industrial applications to quicklyand automatically fabricate arbitrarily complex shapes. SFF processesconstruct shapes by incremental material buildup and fusion ofcross-sectional layers. In these approaches, a three-dimensional (3D)CAD model is first decomposed, or “sliced”, via an automatic processplanner into thin cross-sectional layer representations which aretypically 0.004 to 0.020 of an inch thick. To build the physical shape,each layer is then selectively added or deposited and fused to theprevious layer in an automated fabrication machine. These and other cellcarrier manufacturing techniques are discussed in U.S. Pat. No.6,143,293, the entire contents of which are incorporated herein by thisreference.

In accordance with another aspect of the present invention, the cellcarrier portion may comprise a substance for cellular control, such as achemotactic substance for influencing cell-migration, an inhibitorysubstance for influencing cell-migration, a mitogenic growth factor forinfluencing cell proliferation, a growth factor for influencing celldifferentiation, and factors which promote angiogenesis (formation ofnew blood vessels). Cellular control substances may be located at one ormore predetermined locations on the cell carrier. For example,substances that generally inhibit or otherwise reduce cellular growthand/or differentiation may be located on one surface of the cell carrier(e.g., the surface that will be in proximity to the area within the areaof intended bone formation where bone growth is not desired). Similarly,substances that generally promote or otherwise enhance cellular growthand/or differentiation may be located on one surface of the cell carrier(e.g., the surface that will be in proximity to the area of intendedbone formation). Additionally, the inhibiting and promoting substancesmay be interspersed through the cell carrier at predetermined locationsto help influence rates of cellular growth at different regions over thesurface of the device.

Preferably, an appropriate cell carrier portion of the device will bepre-formed into specific shapes, configurations and sizes that conformto the shape of the cell carrier containment portion by the manufacturer(e.g., by MacroPore Biosurgery, Inc.) before packaging andsterilization. However, a pre-formed cell carrier portion can also beshaped at the time of surgery by bringing the material to its glasstransition temperature, using heating iron, hot air, heated sponge orhot water bath methods. In a certain embodiment, the cell carrier couldbe cut and mechanically press-fit into a containment portion and held inplace by the resulting interference fit. In another embodiment, thecontainment portion could be heated to glass transition temperature torevert to a pre-determined and pre-formed geometry, resulting in aclamping of the cell carrier, resulting in stabilization. In yet anotherembodiment, the cell carrier may be affixed to the containment portionby one or more appropriately sized mating resorbable or non-resorbablescrew, tack or pin inserted through one or more of the apertures in saidcontainment portion. Resorbable screws, tacks or pins can be obtainedfrom MacroPore Biosurgery, Inc. (San Diego, Calif.). Alternatively, onceinserted into an intended area of bone formation, the influx of water tothe area could expand the cell carrier and shrink the containmentportion or shrink the cell carrier and expand the containment portion,resulting in fixation. The cell carrier and the containment portioncould also be affixed to each other using polymer solutions, solvents orappropriate glues.

I. Illustrated Embodiment of a Porous Cell Carrier Portion of the Device

As shown in FIG. 5, a porous cell carrier portion of the device of theinvention 210 is of parabolic shape, having a top face 211, a bottomface 212, side faces 213, a front end 214 and a back end 215, and aplurality of interconnected pores 216.

3. Methods of Adding Regenerative Cells to a Cell Carrier Portion of theDevice

The cell carrier must be appropriately seeded with the regenerativecells to optimize bone formation in the intended area of bone formation.The function of the cell carrier is to deliver cells in a mannercompatible with optimal cell mediated bone formation at the intendedsite of bone formation. The carrier may be a solid, resorbable ornon-resorbable polymer based scaffold with a fiber or spongemacrostructure into which cells are seeded, or a hydrogel, e.g., fibrin,polyethylene glycol, in which cells are encapsulated. The carrier may bemodified by a chemical, peptide, protein, or gene to support cellattachment, e.g., RGD, cell differentiation, e.g., CBFA-1 gene,calcification, e.g., phosphor group. The cells can either be seeded invivo by injection or other appropriate method at the treatment site, orseeded ex vivo.

Ex vivo seeding methods for solid based scaffolds encompassed in thisinvention include art-recognized methods such as static seeding (orcapillary action), injection, dynamic seeding, including uni- andbidirectional perfusion, spinner flask, orbital or random shaker,rotating or end-over-end bioreactor seeding, and vacuum assistedseeding, or a mixture of the methods, to name a few. Seeding methods fornatural or synthetic gel based carrier encompassed in this inventioninclude art-recognized methods such as mixing the cells with the liquidcomponents of the gel based carrier and forming a gel by physicalinteraction crosslinks (e.g. hydrogen, van der waals forces, polarforces, ionic bonds) or covalent crosslinks. Seeding using gel basedcarriers can be accomplished either in vivo or ex vivo. If the cellcarrier portion is of a particulate form, the seeding methods may be asdescribed above, or may be different. For example, the cells may simplybe mixed with the cell carrier portion and place, press fit or injectthe cell/particulate mixture into the intended area of bone formation,such as in the center of the containment device and/or around thecontainment device.

The cells to be incorporated into the carrier may be previouslyincubated or cultured with any substance that may promote the cells'ability to adhere to the carrier or to stimulate the cells' bone formingcapacity, the latter exemplified by bone growth promoting proteins orgenes (e.g.) BMPs, or other bone growth regulatory molecules (e.g.,CBFA-1). In an ex vivo seeding approach, the cell laden cell carriersare then placed into the intended area of bone formation with the cellcarrier containment portion of the present invention to induce boneformation. Once implanted, host cell influx and ADC proliferation,differentiation, osteoid deposition, mineralization, and, in the case ofresorbable scaffolds, concomitant reabsorption of the scaffold,proceeds.

These and other methods of seeding the cell carriers are disclosed in,for example, S. L. Ishaug, G. M. Crane, M. J. Miller, A. W. Yasko, M. J.Yaszemski, and A. G. Mikos. Bone formation by three-dimensional stromalosteoblast culture in biodegradable polymer scaffolds. J Biomed MaterRes 36 (1):17-28, 1997; H. L. Wald, G. Sarakinos, M. D. Lyman, A. G.Mikos, J. P. Vacanti, and R. Langer. Cell seeding in poroustransplantation devices. Biomaterials 14 (4):270-278, 1993; N. S.Dunkelman, M. P. Zimber, R. G. LeBaron, R. Pavelec, M. Kwan, and A. F.Purchio. Cartilage production by rabbit articular chondrocytes onpolyglycolic acid scaffolds in a closed bioreactor system. BiotechnolBioeng 46:299-305, 1995; L. E. Freed, A. P. Hollander, I. Martin, J. R.Barry, R. Langer, and G. Vunjak-Novakovic. Chondrogenesis in acell-polymer-bioreactor system. Exp Cell Res 240 (1):58-65, 1998; R. E.Schreiber, N. S. Dunkelman, G. Naughton, and A. Ratcliffe. A method fortissue engineering of cartilage by cell seeding on bioresorbablescaffolds. Ann N Y Acad Sci 875:398-404, 1999; G. Vunjak-Novakovic, etal (1999) van Wachem PB, tronck JW, oers-Zuideveld R, ijk F, andildevuur CR. Vacuum cell seeding: a new method for the fast applicationof an evenly distributed cell layer on porous vascular grafts.Biomaterials 11 (8):602-606, 1990; L. E. Freed, J. C. Marquis, G.Vunjak-Novakovic, J. Emmanual, and R. Langer. Composition ofcell-polymer cartilage implants. Biotechnol. Bioeng. 43:605-614, 1994;J. L. van Susante, P. Buma, G. J. van Osch, D. Versleyen, P. M. van derKraan, W. B. van der Berg, and G. N. Homming a. Culture of chondrocytesin alginate and collagen carrier gels. Acta Orthop Scand 66 (6):549-56,1995; J. Elisseeff, W. McIntosh, K. Anseth, S. Riley, P. Ragan, and R.Langer. Photoencapsulation of chondrocytes in poly(ethylene oxide)-basedsemi-interpenetrating networks. J. Biomed. Mater. Res. 51:164-171, 2000.

Once seeded on the cell carrier, the device can be inserted into anintended area of bone formation in the recipient. Alternatively, thecells can be seeded onto the cell carrier subsequent to insertion of thedevice (the cell carrier portion and the containment portion) into anintended area of bone formation in the recipient. Once the device isinserted, in-vivo cellular proliferation and, in the case of resorbablecell carriers, concomitant reabsorption of the cell carrier, proceeds.

In accordance with another aspect of the invention, the cell carrierportion may further comprise a substance (e.g., a chemical or biologicalmolecule) to facilitate cell seeding, for example, an adhesion peptideor other biological molecule to enhance cell attachment, a substancethat alters the hydrophilicity of the cell carrier, a surface charge, apolar molecule, or a surfactant or wetting agent to increase cellattachment or increase fluid uptake into the cell carrier. The substanceused to facilitate cell seeding may be located on the surface of thecell carrier pores, the outer regions of the cell carrier, interspersedin the bulk of the cell carrier material, at predetermined locations, orany combinations thereof.

i. Illustrated Embodiment of a Cell Carrier Seeded with RegenerativeCells

As shown in FIG. 17, a porous cell carrier portion of the device of theinvention 210 is in the shape of a parabolic sleeve, having a top face211, a bottom face 212, side faces 213, a front end 214 and a back end215, a plurality of interconnected pores 216 and seeded withregenerative cells such as stem cells and progenitor cells 217.

4. Methods for Manufacturing a Cell Carrier Containment Portion of theDevice

In a broad embodiment, the cell carrier containment portion of thedevice (“the containment portion”) may be constructed from any materialeffective to govern bone formation when used in accordance with themethods disclosed herein. Preferably, the containment portion isresorbable. For example, the containment portion may be constructed fromany biodegradable material, such as resorbable polymers. In preferredembodiments, the containment portion is constructed with materials thatdo not induce a significant antigenic or immunogenic biologicalresponse.

In accordance with one embodiment, non-limiting polymers which may beused to form containment portion of the present invention includepolymers (e.g., copolymers) of lactide (L, D, DL, or combinationsthereof), glycolide, trimethylene carbonate, caprolactone and/orphysical and chemical combinations thereof. In one embodiment, thecontainment portion is comprised of a polylactide, which can be acopolymer of L-lactide and D,L-lactide. For example, the copolymer cancomprise about 60-80% of L-lactide and about 20-40% of D,L-lactide, andin a preferred embodiment the copolymer comprises 70:30 poly(L-lactide-co-D,L-lactide).

In one embodiment, the containment portion is formed by polymers (e.g.,homo and/or copolymers) derived from one or more cyclic esters, such aslactide (i.e., L, D, DL, or combinations thereof), epsilon-caprolactoneand glycolide. For instance, in one embodiment, the containment portioncan comprise about 1 to 99% epsilon-caprolactone, or in anotherembodiment can comprise 20 to 40% epsilon-caprolactone. In one example,a membrane comprises 65:35 poly (L-lactide-co-epsilon-caprolactone). Inother embodiments, butyrolactone, valerolactone, or dimethylpropiolactone can be used with or as a substitute forepsilon-caprolactone. In another embodiment, the containment portion cancomprise a copolymer including lactide and glycolide which is resorbedinto the body more rapidly than the above-mentioned poly(L-lactide-co-D,L-lactide).

The polymers (e.g., co-polymers) of the present invention requirerelatively simple chemical reactions and formulations. The containmentportion of the present invention is preferably smooth and substantiallynon-porous. Preferably, the containment portion may be formed intospecific shapes, configurations and sizes by the manufacturer (e.g.,MacroPore Biosurgery, Inc.) before packaging and sterilization. However,a pre-formed containment portion can also be shaped at the time ofsurgery by bringing the material to its glass transition temperature,using heating iron, hot air, heated sponge or hot water bath methods.

The containment portion may have a thickness of about 0.1 millimeters toabout 5 millimeters. Preferably, the containment portion has a thicknessof about 2 millimeters to about 4 millimeters. The material of thecontainment portion may be flexible enough to conform to a curvature ofa bone and sufficient strength to reduce macro-motion of the bone defectand limit transmission of surrounding motion into the interior spacewhen the device is attached to the targeted area. The containmentportion is adapted for protecting the target area from a prolapse ofadjacent soft tissues into the target area during bone formation and,further, is adapted for preventing resorption of bone due to stressshielding associated with insertion of the device after the desired newbone formation has occurred.

The containment portion may be provided in any shape which mayeffectively promote bone growth. In one embodiment, the containmentportion may be provided in a parabolic shape. In another embodiment, thecontainment portion may be provided in a rectangular shape. In yetanother embodiment, the containment portion may be provided in atrapezoidal shape. In a further embodiment, the containment portion maybe provided in a cylindrical shape. The containment portion of thepresent invention may be sufficiently flexible to conform aroundanatomical structures.

In one embodiment, the containment portion comprises two opposingsurfaces. On one side of the containment portion, there is abone-growing substantially-smooth side or surface, and on the other sidethere is a non-bone growing substantially-smooth side or surface.Preferably, the bone-growing substantially-smooth side is positioned toface the area of intended bone formation, and the non-bone growingsubstantially-smooth side is positioned to face the area where boneformation is not desired.

As used herein, the term “non-porous” refers to a material which isgenerally water tight and, in accordance with a preferred embodiment,not fluid permeable. However, in a modified embodiment of the inventionmicro-pores (i.e., fluid permeable but not cell permeable) may exist inthe containment portion of the present invention, to the extent, forexample, that they do not substantially disrupt the smoothness of thesurfaces of the containment portion to cause scarring of tissue. Insubstantially modified embodiments for limited applications, pores whichare cell permeable but not vessel permeable may be manufactured andused. As presently preferred, the containment portion is manufacturedusing an injection molding procedure to yield a substantially non-porouscontainment portion. The containment portion may also be manufacturedusing extrusion or compression molding procedures. The containmentportion may have a semi-rigid construction, and are fully contourablewhen heated to approximately 55 degrees Celsius.

Accordingly, in particular embodiments, the substantially smooth surfaceof the containment portion may be comprised of a plurality of apertureswith a diameter in a range between 20 microns to 3000 microns. Theapertures are adapted for allowing a proliferation of vasculature andconnective tissue cells therethrough, while preventing the prolapse ofadjacent soft tissues into the biological tissue defect. In addition,the apertures allow vital contributions of blood vessels fromsurrounding tissues, musculature, and periosteum into the target area.Blood vessels invading the target area through the device can greatlyenhance the generation of new bone. The ability for capillaries fromsurrounding tissues to proliferate through the device can help preventmigrating cells from the osseous bed and the periosteum fromoutstripping their proliferating blood supply. This proliferation ofblood vessels can increase the potential of bone formation within agiven target area.

The surface of the containment portion may further be comprised of aplurality of larger apertures with a diameter in a range of 1 to 5millimeters. Such larger apertures in the containment portion arestrategically placed to facilitate insertion of an insertion and/orfixation device for securing the device into an intended area of boneformation. The apertures can also be strategically placed for asubsequent surgical procedure in which additional regenerative cells areinjected through the aperture in the event of a delayed union ornon-union. It is understood in the art that the size of the largerapertures on the containment portion may vary in accordance with thetype of insertion tool and/or fixation device used to secure the devicein the target area. In addition to art-recognized tools for insertion ofthe device, the device may be frictionally secured in the target areausing, fixation devices such as clamps, staples, screws, sutures, tacks,pins and other conventional means. In a preferred embodiment, thefixation devices are resorbable, such as those manufactured by MacroPoreBiosurgery, Inc. In alternative embodiments, a combination of insertiondevices or fixation devices may be used.

It is understood in the art that desired resorption rates will varybased on the particular therapeutic application. It is believed that acontainment portion of the present invention having a thickness of 1millimeters to about 5 millimeters, preferably 2-4 millimeters, shouldmaintain its structural integrity for a period in excess of about onemonth, preferably in excess of about three months, more preferably forabout three to six months, before substantially degrading, so that thebone forming can be achieved and optimized. Similar to the rates ofresorption of the cell carrier portion of the device as set forth above,the rates of resorption of the cell carrier containment portion of thedevice may also be selectively controlled.

Exemplary materials and methods related to making and using all aspectsof the present invention are disclosed in, for example, U.S. Pat. Nos.5,919,234, 6,280,473, 6,269,716, 6,343,531, 6,477,923, 6,391,059,6,531,146 and 6,673,362, the contents of which are incorporated hereinby this reference. Resorbable containment portions of the devices of thepresent invention are available from MacroPore Biosurgery, Inc. (SanDiego, Calif.).

i. Illustrated Embodiment of a Cell Carrier Containment Portion of theDevice

As shown in FIG. 18, in one embodiment, a cell carrier containmentportion of the device of the invention is of parabolic shape 218, havinga top face 219, a bottom face 220, side faces 221, a front end 222 and aback end 223. The surfaces of top and bottom faces 219 and 220 each haveridges 224 to help anchor the containment portion of the device to thesurrounding bone in the target area. The recesses between the ridges 224can enhance mechanical stability that would aid in anchoring thecontainment portion of the device in place. The cell carrier containmentportion 218 may contain apertures 225 of appropriate diameter, that canbe used to screw the device into place. The apertures 225 may also beused to insert an art-recognized tool to insert the device into a targetarea. The cell carrier containment portion has a large center hole 226for insertion of a cell carrier portion of the present invention. FIG.19 depicts an embodiment of the invention wherein a porous cell carrierof parabolic shape has been inserted into a containment portion ofparabolic shape. FIG. 20 depicts an embodiment of the invention whereina porous cell carrier of parabolic shape seeded with the regenerativecells of the invention has been inserted into a containment portion ofparabolic shape. FIG. 20 depicts an embodiment of the device and thecells which is suitable for insertion into a recipient usingart-recognized surgical techniques.

As shown in FIG. 21, in one embodiment, a cell carrier containmentportion of the device of the invention is of trapezoidal shape 227,having a top face 228, a bottom face 229, side faces 230, a front end231 and a back end 232. The surfaces of top and bottom faces 228 and 229each have ridges 233 to help anchor the containment portion of thedevice to the surrounding bone in the target area. The recesses betweenthe ridges 233 can enhance mechanical stability that would aid inanchoring the containment portion of the device in place. The cellcarrier containment portion 227 may contain apertures 234 of appropriatediameter, that can be used to screw the device into place. The apertures234 may also be used to insert an art-recognized tool to insert thedevice into a target area. The cell carrier containment portion has alarge center hole 235 for insertion of a cell carrier portion of thepresent invention.

As shown in FIG. 22, in one embodiment, a cell carrier containmentportion of the device of the invention is of cylindrical shape 236,having a top face 237, a bottom face 238, side faces 239, a front end240 and a back end 241. The surfaces of top and bottom faces 237 and 238each have ridges 242 to help anchor the containment portion of thedevice to the surrounding bone in the target area. The recesses betweenthe ridges 242 can enhance mechanical stability that would aid inanchoring the containment portion of the device in place. The cellcarrier containment portion 236 may contain apertures 243 of appropriatediameter, that can be used to screw the device into place. The apertures243 may also be used to insert an art-recognized tool to insert thedevice into a target area. The cell carrier containment portion has alarge center hole 244 for insertion of a cell carrier portion of thepresent invention.

As shown in FIG. 23, in one embodiment, a cell carrier containmentportion of the device of the invention is of rectangular shape 245,having a top face 246, a bottom face 247, side faces 248, a front end249 and a back end 250. The surfaces of top and bottom faces 246 and 247each have ridges 251 to help anchor the containment portion of thedevice to the surrounding bone in the target area. The recesses betweenthe ridges 251 can enhance mechanical stability that would aid inanchoring the containment portion of the device in place. The cellcarrier containment portion 245 may contain apertures 252 of appropriatediameter, that can be used to screw the device into place. The apertures252 may also be used to insert an art-recognized tool to insert thedevice into a target area. The cell carrier containment portion has alarge center hole 253 for insertion of a cell carrier portion of thepresent invention.

5. Methods for Using the Device for Bone and/or Cartilage Formation

As exemplified herein, the adipose derived regenerative cells of thepresent invention are a rich source of bone or cartilage precursor cellsand they have been shown to form bone and cartilage in vivo.Accordingly, the device of the present invention can be used to treatbone related disorders, e.g., by promoting bone and/or cartilageformation.

Accordingly, in one aspect of the present invention, regenerative cellsare extracted from a donor's adipose tissue, are added to a porousresorbable cell carrier surrounded by a resorbable cell carriercontainment portion, and the combination of the device and the cells isinserted into an intended area of bone formation in a recipient tothereby elicit a therapeutic benefit by promoting bone and/or cartilageformation. In a preferred embodiment the cells are extracted from theadipose tissue of the person into whom they are to be implanted, therebyreducing potential complications associated with antigenic and/orimmunogenic responses to the transplant. Patients are typicallyevaluated to assess bone related disorders by one or more of thefollowing procedures performed by a physician or other clinicalprovider: patient's health history, physical examination, and objectivedata including but not limited to radiographs.

In one embodiment, the harvesting procedure is performed prior to thepatient receiving any products designed to reduce blood clotting inconnection with treatment for the bone related disorder, e.g., spinalfusion surgery. However, in certain embodiments, the patient may havereceived drugs known to affect coagulation (e.g., aspirin) prior to theharvesting procedure. In addition, one preferred method includescollection of adipose tissue prior to any attempted spinal fusionprocedure. However, as understood by persons skilled in the art, thetiming of collection is expected to vary and will depend on severalfactors including, among other things, patient stability, patientcoagulation profile, provider availability, and quality care standards.Ultimately, the timing of collection will be determined by thepractitioner responsible for administering care to the affected patient.

The volume of adipose tissue collected from the patient can vary fromabout 1 cc to about 2000 cc and in some embodiments up to about 3000 cc.The volume of fat removed will vary from patient to patient and willdepend on a number of factors including but not limited to: age, bodyhabitus, coagulation profile, hemodynamic stability, co-morbidities, andphysician preference.

The cells may be extracted in advance and stored in a cryopreservedfashion or they may be extracted at or around the time of defined need.As disclosed herein, the cells may be administered to the patient, orapplied directly to the damaged tissue, or in proximity of the damagedtissue, without further processing or following additional procedures tofurther purify, modify, stimulate, or otherwise change the cells. Forexample, the cells obtained from a patient may be administered to apatient in need thereof via the resorbable cell carrier and resorbablecell carrier containment portion without culturing the cells beforeadministering them to the patient. In one embodiment, the collection ofadipose tissue will be performed at a patient's bedside. Hemodynamicmonitoring may be used to monitor the patient's clinical status.

In accordance with the invention disclosed herein, the regenerativecells can be delivered to the patient soon after harvesting the adiposetissue from the patient. For example, the cells may be administeredimmediately via the device after processing of the adipose tissue andobtaining a composition of regenerative cells. Ultimately, the timing ofdelivery will depend upon patient availability and the processing timerequired to process the adipose tissue. In another embodiment, thetiming for delivery may be relatively longer if the cells to bere-infused to the patient are subject to additional modification,purification, stimulation, or other manipulation, as discussed herein.The number of cells administered to a patient may be related to, forexample, the cell yield after adipose tissue processing. A portion ofthe total number of cells may be retained for later use orcyropreserved. In one embodiment of the invention, a number of cells,e.g., unpurified cells, to be delivered to the patient is expected to beabout 5.5×10⁴ cells. However, this number can be adjusted by orders ofmagnitude to achieve the desired therapeutic effect.

Cells obtained after disaggregation from adipose tissue may be furtherenriched for bone or cartilage progenitor cells by passage over abiologic based component within or separate from the device that eithercaptures chondroprogenitor cells, osteoprecursor cells or all cells butone of these cells. If the former, then an additional step includedwithin or external to the device would be used to release the capturedcells from the biologic based component. An example of thischondroprogenitor or osteoprecursor enrichment would be achromatographic resin to which an antibody that recognizeschondroprogenitor cells or osteoprecursor cells (e.g., SH3 cells) isattached. An example of a release agent is papain or pepsin that couldcleave the antibody/cell complex from the resin.

The cells may also be applied with additives to enhance, control, orotherwise direct the intended therapeutic effect. Cells may beadministered following genetic manipulation such that they express geneproducts that are believed to or are intended to promote the therapeuticresponse(s) provided by the cells. Examples of manipulations includemanipulations to control (increase or decrease) expression of factorspromoting bone or cartilage formation, expression of developmental genespromoting differentiation into specific cartilage or bony cell lineagesor that stimulate cartilage or bony cell growth and proliferation.

The active cell population may be applied to the resorbable cell carrierof the present device alone or in combination with other cells, tissue,tissue fragments, growth factors (e.g., BMPs), or other additiveintended to enhance the delivery, efficacy, tolerability, or function ofthe population. The cell population may also be modified by insertion ofDNA in a plasmid or viral vector or by placement in cell culture in sucha way as to change, enhance, or supplement the function of the cells forderivation of a structural or therapeutic purpose. For example, genetransfer techniques for stem cells are known by persons of ordinaryskill in the art, as disclosed in (Morizono et al., 2003; Mosca et al.,2000), and may include viral transfection techniques, and morespecifically, adeno-associated virus gene transfer techniques, asdisclosed in (Walther and Stein, 2000) and (Athanasopoulos et al.,2000). Non-viral based techniques may also be performed as disclosed in(Muramatsu et al., 1998).

In another aspect, the cells could be combined with a gene encoding apro-chondroprogenitor or pro-osteogenic growth factor(s) which wouldallow cells to act as their own source of growth factor during cartilageor bone formation. Genes encoding anti-apoptotic factors or agents couldalso be applied. Addition of the gene (or combination of genes) could beby any technology known in the art including but not limited toadenoviral transduction, “gene guns,” liposome-mediated transduction,and retrovirus or lentivirus-mediated transduction, plasmid,adeno-associated virus. Cells could be implanted along with a carriermaterial bearing gene delivery vehicle capable of releasing and/orpresenting genes to the cells over time such that transduction cancontinue or be initiated in situ. Particularly when the cells and/ortissue containing the cells are administered to a patient other than thepatient from whom the cells and/or tissue were obtained, one or moreimmunosuppressive agents may be administered to the patient receivingthe cells and/or tissue to reduce, and preferably prevent, rejection ofthe transplant. As used herein, the term “immunosuppressive drug oragent” is intended to include pharmaceutical agents which inhibit orinterfere with normal immune function. Examples of immunosuppressiveagents suitable with the methods disclosed herein include agents thatinhibit T-cell/B-cell costimulation pathways, such as agents thatinterfere with the coupling of T-cells and B-cells via the CTLA4 and B7pathways, as disclosed in U.S. Patent Pub. No. 20020182211. A preferredimmunosuppressive agent is cyclosporine A. Other examples includemyophenylate mofetil, rapamicin, and anti-thymocyte globulin. In oneembodiment, the immunosuppressive drug is administered with at least oneother therapeutic agent. The immunosuppressive drug is administered in aformulation which is compatible with the route of administration and isadministered to a subject at a dosage sufficient to achieve the desiredtherapeutic effect. In another embodiment, the immunosuppressive drug isadministered transiently for a sufficient time to induce tolerance tothe ADC of the invention.

In certain embodiments of the invention, the cells are administered to apatient with one or more cellular differentiation agents, such ascytokines and growth factors. Examples of various cell differentiationagents are disclosed in (Gimble et al., 1995; Lennon et al., 1995;Majumdar et al., 1998; Caplan and Goldberg, 1999; Ohgushi and Caplan,1999; Pittenger et al., 1999; Caplan and Bruder, 2001; Fukuda, 2001;Worster et al., 2001; Zuk et al., 2001).

i. Methods of Using the Device for Intervertebral Disc Repair orInterbody Spinal Fusion Procedures

As set forth herein, back pain remains a major public health problem,especially among aged people. Persistent and severe back pain oftencauses debility and disability. This pain is closely associated withintervertebral disc abnormalities of the spine. By way of background,the human spine is a flexible structure comprised of thirty-threevertebrae. Intervertebral discs separate and cushion adjacent vertebrae,act to minimize loading applied to the spine, and allow motion betweenthe vertebrae (flexion, extension, rotation). An intervertebral disccomprises two major components: the nucleus pulposus and the annulusfibrosis. The nucleus pulposus is centrally located in the disc andoccupies 25-40% of the disc's total cross-sectional area. The matrix ofthe nucleus pulposis is largely composed of a thick gelatinous substancemade up of water, polysaccharides and protein molecules. The annulusfibrosis surrounds the nucleus pulposus and resists torsional andbending force applied to the disc. Vertebral end-plates separate thedisc from the vertebrae on either side of the disc. The outer ring layerstructurally consists mainly of overlapping orthogonal layers offibrocartilage which are mainly made up of type I collagen in anextracellular matrix. This crisscrossing pattern imparts outstandingstructural resilience to the pressure applied by the adjacent vertebrae.The inner core, located in the middle of the annulus fibrosis, consistsof a few cells embedded in a matrix rich in hyaluronic acid and type IIcollagen and acts as a biomechanical shock absorber.

As a result of exertion, injury, illness, accident or abuse, one or moreof the vertebrae and/or one or more discs may become damaged.Specifically, disorders of the vertebrae and discs include but are notlimited to 1) disruption of the disc annulus such as annular fissures;2) chronic inflammation of the disc; 3) localized disc herniations withcontained or escaped extrusions; 4) relative instability of thevertebrae surrounding the disc; and 5) trauma.

Though intervertebral disc cells in both the annulus fibrosus and thenucleus pulposus only constitute a small percent of the disc volume,they have been shown to have the capacity to a synthesize a proteoglycanrich tissue (similar to cartilage) which comprises the extracellularmatrix of the disc. In diseased discs, a decrease in intervertebral disccell density in the nucleus pulposus is accompanied by a reducedproduction of the hydrophilic extracellular matrix. A reduction inintervertebral disc cells thus results progressively in the structuraldecrease of the intervertebral disc space, contributing to degenerativedisc disease (DDD). Traditionally, cartilage is classified as eitherhyaline, elastic, or fibrocartilage. The gel-like anatomicalcharacteristics of the nucleus pulposus (as well as it uniquenotochordal embryonic origin) does not fit into any of the cartilagenouscategories, and is not classically considered a cartilage. It is,nonetheless, permeated with a proteogylcan rich extracellular matrixsimilar to that of cartilage. Based on the ability of the regenerativecells of the present invention to produce cartilage and bone, thesecells can also stimulate the formation of the cartilage likeproteoglycan rich extracellular matrix to thereby restore theintervertebral disc.

Various approaches have been developed to treat back pain. Minor backpain can be treated with medication and other non-invasive therapy. Mildto moderate degenerated discs may be treated by restoring the damagestissues within the disc, nucleous pulposis and annulus fibrosis.Accordingly, the regenerative cells of the invention may be used tostimulate cartilage development and thereby restore the intervertabraldiscs at various stages of degeneration. For example, the regenerativecells of the invention may be delivered via injection in a physiologicsolution directly into the disc's nucleus pulposis in cases of milddegeneration, wherein the disc is largely still grossly intact, butbeginning to break down. In cases of moderate disc degeneration, wherethe disc is macroscopically degenerating, the regenerative cells couldbe mixed with a hydrogel liquid that gels in situ when injected into thenucleus pulposis. The gelation could be thermal or thermochemicallymediated.

However, it is often necessary to remove at least a portion of thedamaged and/or malfunctioning back component. For example, when a discbecomes ruptured, a discectomy surgical procedure can be performed toremove the ruptured disc and to fuse the two vertebrae between theremoved disc together. Details regarding typical implementations ofmethods for fusing vertebrae are disclosed in U.S. Pat. Nos. 6,033,438and 5,015,247, the contents of which are incorporated in theirentireties herein by reference.

Spinal fusion is indicated to provide stabilization of the spinal columnfor disorders such as structural deformity, traumatic instability,degenerative instability, and post resection iatrogenic instability.Fusion, or arthrodesis, can thus be achieved, for example, by theformation of an osseous bridge between adjacent motion segments. Thefusion can be accomplished either anteriorly between contiguousvertebral bodies or posteriorly between consecutive transverseprocesses, laminae or other posterior aspects of the vertebrae. Surgicaltechniques for spinal fusion are well-known in the art. The presentinvention provides an inherent advantage over the art-recognizedtechniques because it eliminates the need for a second surgery toharvest autologous bone graft. In addition, typically, bone graftmaterials and/or growth factors are often needed to promote spinalfusions. The present invention provides autologous regenerative cellsthat promote bone growth cost-effectively and without fear of graftrejection.

Surgical methods for spinal fusion and other surgical techniques fortreating bone related disorders are well known to the ordinarily skilledartisan. All such art recognized techniques are intended to beencompassed by the present invention.

The present invention is further illustrated by the following exampleswhich in no way should be construed as being further limiting. Thecontents of all cited references, including literature references,issued patents, published patent applications, and co-pending patentapplications, cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES

The ADC or regenerative cells used throughout the examples set forthbelow can be obtained by the method(s) described in the instantdisclosure and/or the method(s) described in U.S. application Ser. No.10/316,127, entitled SYSTEMS AND METHODS FOR TREATING PATIENTS WITHPROCESSED LIPOASPIRATE CELLS, filed Dec. 9, 2002, which claims priorityto U.S. Provisional Application Ser. No. 60/338,856, filed Dec. 7, 2001,as well as well as the methods described in U.S. application Ser. No.10/887,822 entitled, SYSTEMS AND METHODS FOR SEPARATING ANDCONCENTRATING REGENERATIVE CELLS FROM TISSUE, filed Jun. 25, 2004, whichclaims priority to U.S. application Ser. No. 10/316,127, entitledSYSTEMS AND METHODS FOR TREATING PATIENTS WITH PROCESSED LIPOASPIRATECELLS, filed Dec. 9, 2002, which are all commonly assigned and thecontents of all of which are expressly incorporated herein by thisreference.

Example 1 Human Adipose Tissue is a Source of Osteoprogenitor CellsMethods and Materials:

Liposuction: Human adipose tissue was obtained from individualsundergoing elective lipoplasty (liposuction). In general the liposuctionprocedure involved creation of a 0.5 cm incision in the skin of theabdomen and/or thigh followed by infiltration of the subcutaneous spacewith tumescent solution (saline supplemented with lidocaine andepinephrine). After approximately 10 minutes the subcutaneous adiposetissue was aspirated through a cannula (3-5 mm diameter according toPhysician preference) under machine-assisted suction. Adipose tissue,blood, and tumescent solution (lipoaspirate) was collected into adisposable container and stored at 4° C. during transport to thelaboratory.

Tissue Digestion: Adipose tissue was washed several times with warm (37°C.) saline to remove excess blood and free lipid. The tissue was thendigested at 37° C. with Blendzyme collagenase (Blendzyme 1 for 45minutes or Blendzyme 3 for 20 minutes; Roche, Indianapolis, Ind.) inapproximately two volumes of saline. Constant agitation was applied toensure adequate mixing of tissue and medium throughout the digestion.After digestion cells non-buoyant cells were removed and washed threetimes to remove residual enzyme. Cells were counted using a vital dye(propidium iodide:) on a hemocytometer using a fluorescence microscope

CFU-AP Assay: Washed cells plated in duplicate at 1,000, 5,000, and25,000 cells/well in six multiwell plates (Corning, N.Y.). Osteogenicdifferentiation was induced by culturing cells for 3 weeks in mediumconsisting of α-MEM (Cellgro, Herndon, Va.), 10% FBS/5% Horse serum/1%Antibiotic-antimycotic solution (Omega Scientific, Tarzana, Calif.)supplemented with 0.1 μM Dexamethasone (Sigma, St. Louis, Mo.), 10 mMβ-glycerophosphate (Sigma, St. Louis, Mo.) and 50 μM L-Ascorbic acid2-phosphate (Sigma, St. Louis, Mo.). After the first week of inductionthe culture media was changed weekly with Osteogenic Medium withoutdexamethasone. To detect Alkaline Phosphatase (AP) activity, cultureplates were rinsed with PBS and stained with 1% Napthol AS-BI phosphate(Sigma, St. Louis, Mo.) and 1 mg/ml Fast Red TR (Sigma, St. Louis, Mo.)for 20 min at 37° C. After staining, the cells were washed with PBS andfixed with 10% Buffered Formalin Phosphate (Fisher Scientific,Pittsburgh, Pa.) for 15 min. Colonies consisting of more than 50 cellswith positive AP staining were defined as CFU-AP. The number of CFU-APwas counted in each well and the average number of CFU-AP from theduplicate sample was calculated.

Alkaline Phosphatase Assay: Total alkaline phosphatase activity in day21 cultures of adipose tissue-derived cells was measured using acolorimetric assay (Sigma Diagnostics, St. Louis, Mo.). Briefly, day 21cultures were harvested by scraping, cells were resuspended in saline,and lysed by sonication. Cell lysates were then incubated withp-nitropheyl phosphate at pH 10.2 for 15 minutes at 30° C. Absorbance at405 nm was determined using a Turner SP-830 spectrophotometer (TurnerDesigns, Sunnyvale, Calif.).

Results:

Donor Characteristics: The characteristics of the donor population isshown in Table A below. This population is representative of thepopulation undergoing cosmetic lipoplasty. Data from the AmericanSociety for Aesthetic Plastic Surgery indicate that 384,626 liposuctionprocedures were performed in the USA in 2003 making this the most commoncosmetic surgery procedure for both men and women. Women accounted for84% of all lipoplasty procedures (81% in the present study).Approximately half of all patients undergoing lipoplasty in 2003 werebetween 35 and 50 years of age; median age in this study 46.

TABLE A Donor Characteristics Gender 44 F  10 M Median Age 46 (range19-72) Mean Volume of Lipoaspirate 510 ± 47 ml (range 50-1,500) Timefrom Liposuction to Tissue Processing <6 hours 17 Overnight 37 Body MassIndex Normal 20 Overweight 12 Obese 6 Unknown 16

Development and Validation of the CFU-AP Assay: Tissue processingyielded an average of 7.3±0.1×10⁵ viable non-buoyant nucleated cells perunit volume (ml) of adipose tissue. These cells were then plated inCFU-AP assays at multiple densities in order to validate this assay forthis cell source. Further, we have demonstrated that CFU-AP count islinear with cell number plated across the range of 1,000 to 50,000cells/well. The range of frequency detected is such that for standardassays we routinely plate cells in triplicate at two different cellconcentrations in order to ensure that we have cultures in which colonynumber per well falls into the range of 10-60 per well.

Osteoprogenitor Frequency and Yield

Using this approach we tested adipose tissue obtained from 54 donors(Table A). The data shows that CFU-AP frequency in samples processedfrom fresh tissue (tissue processed within six hours of collection) wassignificantly higher than that of samples processed on the day followingtissue collection (2.0±0.3 CFU-AP/100 cells compared with 0.6±0.2CFU-AP/100 cells; p=0.001). CFU-AP frequency did not correlate withdonor age or gender.

The average yield of osteoprogenitor cells was 7,225±1,760 CFU-AP/mladipose tissue (range 120-67,500). Unlike CFU-AP frequency there was atrend towards reduced CFU-AP yield per unit volume of adipose tissuewith increasing donor age. However, when the upper quartile of age (>48yr) was compared with the lower age quartile (<34 yrs) the differencedid not reach statistical significance (4,300±1,500 cells/ml tissue forolder donors compared to 9,200±1,800 for younger donors; p=0.09).

Alkaline Phosphatase Activity

In this study a colony was only counted for the purposes of the assay ifit was alkaline phosphatase-positive and contained at least 50 cells.Colony size varied considerably with some colonies consisting of severalthousand cells. Total alkaline phosphatase activity might represent asuperior way to measure osteogenic activity as it takes intoconsideration both total osteoblastic cell number (a function ofprogenitor cell proliferation) and the degree of differentiation ofthese cells. Thus, alkaline phosphatase assays were performed inreplicate wells in 15 consecutive donors. Total alkaline phosphataseactivity exhibited a linear relationship with cell number plated. Whenalkaline phosphatase activity (normalized to cell number plated) wasthen plotted against age the data demonstrate an inverse relationshipbetween age and alkaline phosphatase activity (FIG. 2; r^(2=0.57)).

Osteoprogenitor Cell Yield and Body Mass Index

Data on donor height and weight were available for 38 donors. Thisallowed calculation of Body Mass Index (BMI) and stratification intothree groups; 20 donors of normal BMI (between 18.5 and 24.9), 12 in theOverweight category (25.0-25.9), and 6 Obese donors (≧30). The yield ofCFU-AP per unit volume of tissue was significantly higher in persons ofnormal BMI than for those who were overweight (5,279±780 in normal BMIdonors versus 1,373±1,150 in overweight donors; p<0.02). Obese donorsshowed considerable variation in CFU-AP yield per unit volume of tissue(867-18,462 CFU-AP/ml; FIG. 3). The average was higher than foroverweight donors but this did not reach statistical significance(p=0.08).

SUMMARY

The foregoing results indicate that human adipose tissue may be a richsource of osteprogenitor cells. Specifically, the results indicate thathuman adipose contains an average of approximately 7,200 osteoprogenitorcells per gram of tissue. The frequency of CFU-AP within this populationis depended upon the time from tissue collection to tissue processingsuch that tissue processed within six hours of collection yielded afrequency of 2.0±0.3 CFU-AP/100 nucleated cells. Studies with freshlyharvested human bone marrow have estimated CFU-AP yield at 200-2,000CFU-AP/ml marrow at a frequency of approximately 1 in 50,000 (Banfi,2001; Galotto, 1999; D'Ippolito 1999) Thus, this data indicates thathuman adipose tissue contains substantially more osteoprogenitor cellsthan a similar volume of marrow.

Adipose tissue utilized for the foregoing study was obtained usingliposuction procedures which are optimized for tissue extraction and notfor recovery of viable cells. However, using the systems and methods ofthe present invention, in combination with altered parameters forliposuction (i.e., aimed at recovery of viable cells by for examplealtering the amount of suction force applied and the style and size ofthe cannula used) will undoubtedly result in an even greaterregenerative cell yield. Higher osteoprogenitor cell yield isparticularly important in the context of the device of the presentinvention. For example, a higher frequency of osteoprogenitor cells willallow application of a higher dose and potentially greater clinicalefficiency in promoting bone formation.

Example 2 Adipose Derived Regenerative Cells Form Cartilage and Bone InVivo Methods and Materials: Cell Scaffolds: PLA Scaffold Fabrication andPreparation:

Porous polymer scaffolds having an approximate porosity of 90% weremanufactured by a solvent-casting/particulate-leaching method (see e.g.,A. G. Mikos, A. J. Thorsen, L. A. Czerwonka, Bao Y., and R. Langer.Preparation and characterization of poly(L-lactic acid) foams. Polymer35 (5):1068-1077, 1994.). Briefly, sodium chloride was used as theleachable porogen and was sieved into particles ranging in diameter of300-500 p.m. A 3% polymer solution was made by combining 70:30poly(L-lactide-co-DL-lactide) with chloroform. The sieved salt andpolymer solution were mixed in Teflon Petri dishes to make a 9:1 weightratio of salt to polymer and the solvent was allowed to evaporate in thehood for 2 days. The salt was leached from the scaffold by immersion inwater for 3 days, with water changes every 8-12 hours, Residual solventwas removed by placing the porous polymeric sponges under low vacuum for2 days. Scaffolds were sized for implantation by punching 8 mm diameterimplants from the polymer sheets. Scaffolds were rendered aseptic byovernight submersion in 70% ethanol and exposure to ultraviolet light,rinsed 3 times with 0.9% injectable saline to, then the residual salinewas removed from the scaffold prior to cell seeding.

Demineralized Bone Matrix (DBM) Scaffold Preparation and Cell Seeding:

Grafton® Demineralized Bone Matrix Putty (Osteotech, N.J.) implants wereprepared for cell seeding by rinsing evenly divided portions(approximately 70 mg per implant) with 0.9% injectable saline, 3 times,prior to removal of residual saline from the scaffold.

Adipose Derived Regenerative Cell Isolation, Culture, and Cell Seeding:

ADCs were obtained by excising the white subcutaneous adipose tissuefrom transgenic mice expressing Green Fluorescent Protein[C57B1/6-TgN(ACTbEGFP)1Osb]. Tissue was subjected to enzymaticdigestion. Briefly, cells were washed in 1×PBS and digested in 0.075%Collagenase I (Sigma) for 45 min at 37° C. shaking, then neutralizedwith 10% FBS (Fisher) in Dulbecco's Modified Essential Medium (Gibco),passed through a gradient of cell straining filters (100 μm, 70 μm and40 μm), and centrifuged at 400 g for 5 min. The cell pellet wasresusupended in osteogenic culture medium [α-MEM (Cellgro, Herndon,Va.), 10% FBS, 1% Antibiotic-antimycotic solution (Omega Scientific,Tarzana, Calif.) supplemented with 0.1 μM Dexamethasone (Sigma, St.Louis, Mo.), 10 mM J3-glycerophosphate (Sigma, St. Louis, Mo.) and 50 μML-Ascorbic acid 2-phosphate (Sigma, St. Louis, Mo.)]. After the first 3days of induction the culture media was changed weekly with osteogenicmedium without dexamethasone. Flasks were passaged twice in a 3 weekculture period. Cultured ADCs were then incubated overnight withosteogenic medium in the presence or absence of recombinant human BMP-2(5 μg/ml). Cells were then resuspended in 0.9% injectable saline (5×10⁷cells/ml) and kept at 4° C. until seeding onto PLA or Grafton DBMscaffolds at 2.5×10⁶ cells per scaffold.

Surgical Procedure:

Athymic rats (approximately 200 g) were anesthetized withketamine/xylazine [100/10 mg/kg (TW Medical Veterinary Supply),respectively]. The rats' abdomens were shaven, swabbed with betadine and70% isopropanol. Under sterile conditions, a full-thickness incision,approximately 1.5 cm in length through the skin of the leg close to thehip area was made. The tensor muscle was exposed via blunt dissectionand a 1.0-cm incision along the tensor muscle was made, dissecting thefascia lata to expose the rectus muscle. Using a microspatula, a channelwas then created, into which a single implant was placed. A single passof 5-0 silk sutures were then used to close off the channel, followed bysuturing of the tensor muscle incision with 5-0 coated Vicryl® braidedsuture, and closure of the skin incision with 9-mm wound clips.Following surgeries, allow animals recovered from anesthesia on heatingpads. Buprenorphine (0.02-0.05 mg/kg, subcutaneously) was administeredupon awakening, in order to relieve post-surgical pain.

Analysis:

Two weeks after surgery, rats were euthanized via CO₂ exposure, andimplants were recovered. Tissues were fixed overnight in 0.4%paraformaldehyde, embedded in paraffin, cut into 5 micron sections, thenstained with Toluidine Blue or Von Kossa to visualize cartilage andmineralized tissue, respectively. Unstained sections were evaluated forthe presence of green fluorescent donor cells.

Results:

Tissues implanted with osteoconductive (DBM) or non-osteoconductive(PLA) scaffold seeded with ADCs that had been primed ex situ with BMP-2contained a mixture of cartilage, osteoid, and mineralized bone.Cartilage was identified in Toluidine Blue stained sections by thepresence of metachromatically stained cells and tissue. Chondrocyteswere present within lacunae. These were surrounded by aproteoglycan-rich matrix. Osteoid was evident as a smooth matrix inwhich osteocytes were embedded. Mineralized bone was evident in VonKossa stained sections. Early hematopoietic elements were identified inbone by the presence of cell laden medullary cavities. Cartilage wasalso evident in tissues with implants composed of unprimed ADCs and DBM.In contrast, no cartilage or bone was present on either scaffold typeimplanted without ADCs.

SUMMARY

In summary, the foregoing results demonstrate that adipose derivedregenerative cells have the capacity to form cartilage and bone whendelivered into skeletal muscle on an osteoconductive (DBM) ornon-osteoconductive (PLA) scaffold. Thus, adipose derived regenerativecells are in stimulating cartilage formation to restore theintervertebral disc. Moreover, the foregoing study supports the use ofadipose derived regenerative cells in stimulating bone formation in bothintervertebral body and intertransverse process spine fusion, bothenvironments of which are surrounded by skeletal muscle. It is likelythat the skeletal muscle, which contains satellite cells that are knownosteoprogenitors, act in concert with adipose derived regenerative cellsto stimulate robust bone formation. At a minimum, skeletal muscleprovides a supportive environment in which adipose derived regenerativecells can stimulate bone formation.

Example 3 Regenerative Cells Form Cartilage Bone and Skeletal Muscle InVitro Materials and Methods: Regenerative Cell Preparation

Human regenerative cells were harvested by enzymatic digestion asdescribed in herein. Isolated regenerative cells were then separatedinto CD34 (+) and CD34 (−) populations using immuno magnetic beads. Eachsub-population of cells were cultured in complete medium (DMEM, 10%fetal bovine serum, 5% horse serum, and 1% antibiotic, antimycoticsolution) at multiple densities in multi-well culture plates. At 3weeks, CD34 (−) clones were selected from these cultures and subculturedfor approximately 2 months in one of the following culture mediums:complete medium, osteogenic medium, or chondrogenic medium.

Analysis:

Differentiation of clonal cells towards cartilage or bone was determinedby staining with alcian blue or alkaline phosphatase, respectively.Differentiation of clonal cells towards muscle was assessed byimmunohistochemical staining of CD34 (+) and CD34 (−) cells usingantibodies directed towards human myosin heavy chain, as well as theskeletal muscle specific markers myf5 and myoD1. RT-PCR was performed toassess gene expression of myosin heavy chain, myf5, and myoD1.

Results:

Regenerative cells from CD34 (−) clonal colonies differentiated tochondrocytes, osteoblasts, and muscle cells, depending on the culturemedium used. regenerative cells cultured in osteogenic mediumdifferentiated to osteoblasts, while regenerative cells cultured inchondrogenic medium differentiated to chondrocytes, as evidenced byhistochemical staining with alcian blue and alkaline phosphatase,respectively. Moreover, regenerative cells from CD34 (−) clonal coloniesthat were cultured in complete medium in the absence of anydifferentiation medium were immunohistochemically positive for myosinheavy chain, MyfS and MyoD, demonstrating their differentiation to amyoblast phenotype. Consistent with the immunohistochemical data, thesemuscle clones expressed the genes for myosin heavy chain, MyfS, andMyoD.

Summary:

In summary, these data show that human cultured regenerative cellcolonies give rise to chondrocytes, osteoblasts, and skeletal myoblasts.Thus, the regenerative cells of the present invention have the abilityto stimulate cartilage, bone, and skeletal muscle formation to restorelost tissue, e.g., soft tissue tears in the cartilage, tendon, orligament, as well as in repairing hard tissue breaks, such as innon-union long bone fractures. Additionally, the regenerative cells ofthe present invention have the ability to generate tissue de novo inorder to impart mechanical support, e.g., in spinal fusion applicationsby bone formation between the transverse processes or vertebrae.

Any feature or combination of features described herein are includedwithin the scope of the present invention provided that the featuresincluded in any such combination are not mutually inconsistent as willbe apparent from the context, this specification, and the knowledge ofone of ordinary skill in the art. For purposes of summarizing thepresent invention, certain aspects, advantages and novel features of thepresent invention have been described herein. Of course, it is to beunderstood that not necessarily all such aspects, advantages or featureswill be embodied in any particular embodiment of the present invention.Additional advantages and aspects of the present invention are apparentin the following detailed description and claims.

The above-described embodiments have been provided by way of example,and the present invention is not limited to these examples. Multiplevariations and modification to the disclosed embodiments will occur, tothe extent not mutually exclusive, to those skilled in the art uponconsideration of the foregoing description. Additionally, othercombinations, omissions, substitutions and modifications will beapparent to the skilled artisan in view of the disclosure herein.Accordingly, the present invention is not intended to be limited by thedisclosed embodiments, but is to be defined by reference to the appendedclaims.

A number of publications and patents have been cited hereinabove. Eachof the cited publications and patents are hereby incorporated byreference in their entireties.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of restoring cartilage, tendon or ligament in a patient,comprising: a) providing a tissue removal system and a prosthetic devicecomprising a cell carrier portion and a cell carrier containmentportion, said cell carrier containment portion being configured to atleast partially contain the cell carrier portion; b) removing adiposetissue from the patient using the tissue removal system; c)cryopreserving the adipose tissue; c) processing the cryopreservedadipose tissue to obtain a concentrated population of cells comprisingadipose-derived stem cells; d) introducing the concentrated populationof cells comprising adipose-derived stem cells to the cell carrierportion of the prosthetic device; and e) inserting the prosthetic devicecontaining the concentrated population of cells comprisingadipose-derived stem cells into the intended bone formation area in thepatient.
 2. The method of claim 1, wherein cartilage, tendon or ligamentis restored in the patient.
 3. A method of restoring cartilage, tendonor ligament in a patient comprising: a) providing a self containedadipose derived stem cell processing unit and a device comprising a cellcarrier portion and a cell carrier containment portion, wherein saidcell carrier containment portion is configured to at least partiallycontain the cell carrier portion, wherein said self contained adiposederived stem cell processing unit comprises a tissue collection chamberthat is configured to receive unprocessed adipose tissue that is removedfrom said patient, wherein the tissue collection chamber is defined by aclosed system; a first filter that is disposed within said tissuecollection chamber, wherein said first filter is configured tosubstantially retain a first component of said unprocessed adiposetissue and substantially pass a second component of said unprocessedadipose tissue, such that said first filter substantially separates saidfirst component from said second component, and wherein said firstcomponent comprises a cell population comprising adipose-derived stemcells and said second component comprises lipid and mature adipocytes; aprocessing chamber, which is configured to receive said first componentcomprising said population of cells comprising adipose-derived stemcells from said tissue collection chamber, wherein said processingchamber is within said closed system; a conduit configured to allowpassage of said first component comprising said cell populationcomprising adipose-derived stem cells from said tissue collectionchamber to said processing chamber while maintaining a closed system; acell concentrator disposed within said processing chamber, which isconfigured to facilitate the concentration of said first componentcomprising said cell population comprising adipose-derived stem cells soas to obtain a concentrated population of cells comprisingadipose-derived stem cells, wherein said cell concentrator comprises acentrifuge or a filter; and an outlet configured to allow the asepticremoval of said concentrated population of cells comprisingadipose-derived stem cells; b) removing adipose tissue from the patientusing the tissue removal system; c) crypreserving the adipose tissue; d)thawing the adipose tissue; e) processing the adipose tissue to obtain aconcentrated population of cells comprising adipose-derived stem cells;f) inserting the device into the intended cartilage, tendon or ligamentrestoring area in the patient; and g) introducing the regenerative cellsto the cell carrier portion of the device inserted into the patient. 4.The method of claim 3, wherein introducing the concentrated populationof cell comprising adipose-derived stem cells to said cell carrierportion of the device inserted into the patient restores cartilage,tendon or ligament in the patient.
 5. The method of claim 3, wherein theself-contained adipose-derived stem cell processing unit furthercomprises a programmable processing device capable of communicating withand controlling the tissue collection chamber and the cell concentrator.6. The method of claim 3, wherein the self contained adipose-derivedstem cell processing unit further comprises a user interface for a userto input parameters into the system.
 7. The method of claim 3, whereinthe self contained adipose-derived stem cell processing unit furthercomprises a display screen to display instructions that guide a user toinput parameters, confirm pre-programmed steps, or warn of errors orcombinations thereof.